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U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— Bulletin  137. 

A.  D.  MELVIN,  Chief  of  Burrau. 


ANTHRAX, 


SPECIAL  REFERENCE  TO  THE  PRODUCTION 
OF  IMMUNITY. 


BY 

CHARLES  F.  DAWSON,  M.  D.,  D.  V.  S., 
Veterinarian,  Delaware  College  Agricultural  Experiment  Station, 

UNDER  THE  DIRECTION  OF 

JOHN  R.  MOHLER,  V.  M.  D., 

Chief  of  the  Pathological  Division, 

Bureau  of  Animal  Industry. 


'alifornia 
?ional 


WASHINGTON: 

GOVERNMENT   PRINTING   OFFICE. 

1911. 


Digitized  by  tine  Internet  Archive 

in  2007 -witii  funding  from 

IVIicrosoft  Corporation 


littp://www.archive.org/details/antliraxwitlispeciOOdawsiala 


Issued  June  12, 1911. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— Bulletin  137. 
A.  D.  MELVIN,  Chief  op  Burbau. 


ANTHRAX, 


WITH  SPECIAL  REFERENCE  TO  THE  PRODUCTION 

OF  IMMUNITY. 


BY 

CHARLES  F.  DAWSON,  M.  D.,  D.  V.  S., 
VeterinatHan ,  Delaware  College  Agricultural  Experiment  Station^ 

UNDER  THE  DIRECTION  OF 

JOHN  R.  MOHLER,  V.  M.  D., 

Chief  of  the  Pathological  Division, 

Bureau  of  Animal  Industry. 


WASHINGTON: 

GOVERNMENT   PRINTING   OFFICE. 

1911. 


THE  BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  Melvin. 
As»istant  Chief:  A,  M.  Fabbington. 
Chief  Clerk:  Chables  C.  Cabboll. 

Animal  Husbandry  Division:  Geobge  M.  Rommel,  chief. 
Biochemic  Division:  M.  Dorset,  chief. 
Dairy  Division:  B.  H.  Rawl,  chief. 

Inspection  Division:  Rice  P.  Steddom,  chief;  Morbis  Wooden,  R.  A.  Ramsay, 
and  Albert  E.  Behnke,  associate  chiefs. 
Pathological  Division:  John  R.  Mohler,  chief. 
Quarantine  Division:  Richabd  W.  Hickman,  chief. 
Zoological  Division:  B.  H.  Ransom,  chief. 
Experiment  Division:  E.  C.  Schroedeb,  superintendent. 
Editor:  James  M.  Pickens. 


LETTER  OF  TRANSMITTAL 


U.  S.  Department  of  Agriculture, 

Bureau  or  Animal  Indusihy, 

Washington,  D.  C,  March  20, 1911. 
Sir  :  I  have  the  honor  to  transmit  for  publication  the  accompanying 
manuscript  on  the  subject  of  anthrax,  by  Dr.  Charles  F.  Dawson,  of 
the  Delaware  College  Agricultural  Experiment  Station,  the  experi- 
mental work  having  been  carried  out  in  cooperation  with  the  Patho- 
logical Division  of  this  bureau. 

Anthrax  is  one  of  the  oldest  and  most  widely  distributed  diseases  of 
animals,  and  is  particularly  destructive  to  cattle  and  sheep.  The 
existing  method  of  combating  the  infection  is  to  endeavor  to  control 
it  by  means  of  a  vaccine  devised  by  Pasteur.  Two  injections  are, 
however,  necessary  by  this  method,  and  an  active  immunity  is  not 
established  for  about  four  weeks. 

Two  important  results  ha^'e  been  accomplished  through  the  work 
described  in  this  paper.  A  single  vaccine  has  been  produced  which 
may  be  substituted  for  the  above-mentioned  double  vaccine,  thus  cut- 
ting down  the  time  one-half.  The  other  result  is  the  production  of 
an  antianthrax  serum  that  will  confer  an  immediate  passive  immu- 
nity. Thus  the  tAvo  combined  are  a  distinct  advantage  over  the  exist- 
ing method  in  the  case  of  sudden  outbreaks,  where  there  is  no  time  to 
prepare  for  the  disease  in  the  usual  way.  This  serum  can  also  be  used 
in  conjunction  with  the  double  vaccine  of  Pasteur. 

I  recommend  that  the  paper  be  published  in  the  bulletin  series  of 
this  bureau. 

Eespectfully,  A.  D.  jNIelvin, 

Chief  of  Bureau,. 
Hon.  James  Wilson, 

Secretary  of  Agriculture. 

3 


CONTENTS. 


Page. 

Introduction 5 

Historical 5 

The  anthrax  bacillus 6 

Cultural  and  morphological  characters 7 

Channels  of  infection 9 

Infection  of  the  soil 10 

Methods  of  diagnosis 11 

Preparation  of  specimens 11 

Animal  inoculations 12 

Different  forms  of  anthrax 13 

Symptoms 14 

Post-mortem  appearances 15 

The  cause  of  death 15 

Disposal  of  carcasses Ig 

The  official  method  in  Delaware 17 

Susceptibility  of  the  various  animals 17 

Anthrax  in  man 19 

Malignant  pusttde 19 

Pulmonary  and  intestinal  anthrax 20 

The  anthrax  season 20 

Prevalence  of  anthrax  in  Delaware 22 

Methods  of  producing  immunity  in  animals 24 

The  existing  methods  of  double  vaccination  (Pasteur  method) 24 

Effectiveness  of  the  method 25 

A  test  of  Pasteur  vaccines 26 

Experiments  with  various  substances  to  test  immunizing  powers 28 

Anthraxin 28 

Pyocyanase 30 

Anthraxase 35 

Anthraxoin 38 

A  commercial  vaccine  in  pill  form 40 

Preparation  of  an  effective  single  vaccine 41 

Tests  of  the  single  vaccine 42 

A  serum  for  producing  immediate  immunity 43 

Experiments  with  the  serum 44 

Conclusion 47 

4 


ANTHRAX, 

WITH  SPECIAL  REFERENCE  TO  THE  PRODUCTION  OF  IMMUNITY. 


INTRODUCTION. 

Anthrax  is  primarily  a  disease  of  herbivorous  animals,  and  occurs 
as  an  epizootic  in  sheep,  cattle,  horses,  and  mules,  the  animals  being 
susceptible  in  the  order  named.  In  experimental  work  the  small  ani- 
mals, such  as  mice,  guinea  pigs,  and  rabbits,  are  extremely  suscep- 
tible. The  French  name  for  the  disease  is  "  charbon ;  "  the  German, 
'•■  Milzbrand."  When  it  occurs  in  domestic  animals  it  is  also  some- 
times known  as  splenic  fever.  In  natural  outbreaks  anthrax  usually 
has  the  character  of  a  rapidly  fatal  septicemia,  with  the  presence  of 
large  numbers  of  the  characteristic  bacilli  in  the  blood. 

IVliile  the  disease  does  not  occur  as  a  natural  infection  in  man  in 
the  same  sense  as  in  animals,  man  frequently  becomes  infected  from 
handling  infected  animals  or  their  products,  such  as  hides  and  hair. 
When  man  becomes  infected  through  abrasions  of  the  skin,  the  result- 
ing disease  process  is  carbunculous  in  nature  and  is  known  as  "  malig- 
nant pustule."  When  the  infection  takes  place  in  the  lungs,  the  dis- 
ease is  known  under  the  name  "  woolsorters'  disease."  A  third  form 
in  man  may  attack  the  intestinal  tract.  The  latter  two  forms  are 
rapidly  fatal.  In  malignant  pustule  the  disease  may  remain  localized 
and  the  patient  may  recover.  If,  however,  the  bacilli  migrate  from 
the  local  lesion  to  the  blood  stream,  death  rapidly  ensues,  the  patient 
dying  from  septicemia  or  toxemia. 

HISTORICAL. 

Anthrax  is  one  of  the  oldest  diseases.     Moses  records  it  in  Exodus 

9 : 9.     Homer,  Ovid,  Plutarch,  Virgil,  Pliny,  and  many  others  have 

mentioned  it  in  their  writing.     It  exists  in  all  countries  and  in  all 

latitudes.     It  was  formerly  very  destructive  to  human  life  as  well 

as  to  animals.     In  1G17,  near  Naples,  G0,000  people  arc  reported  to 

have  died  from  anthrax.     In  San  Domingo,  in  1770,  15,000  persons 

perished  in  the  short  period  of  six  weclvs.     This  enormous  death  rate 

was  probably  due,  in  part,  to  eating  the  carcasses  of  animals  dead 

from  the  disease. 

5 


D  ANTHRAX. 

It  was  not  until  the  middle  of  the  nineteenth  century  that  scientists 
began  the  serious  study  of  this  very  destructive  disease.  In  1849, 
Pollender,  while  studying  the  blood  of  animals  dead  from  anthrax, 
discovered  numerous  rod-shaped,  microscopic  bodies,  which  he 
claimed  were  the  cause  of  the  disease.  This  claim  remained  unsub- 
stantiated until  18G3,  when  Davaine  announced  that  the  bodies  dis- 
covered by  Pollender  were  bacteria,  and  showed  that  the  blood  of  an 
animal  could  not  cause  anthrax  in  another  animal  when  injected  into 
it  unless  it  contained  these  bodies.  To  this  bacterium  Davaine  gave 
the  present  name,  Bacillus  anthracis.  Davaine's  views  were  not 
immediately  accepted,  but  others  took  up  the  work,  and  in  1876  Dr. 
Robert  Koch,  in  making  his  first  contribution  to  bacteriology, 
announced  that  he  had  been  successful  in  proving  the  correctness  of 
Davaine's  work  on  anthrax,  and  the  question  was  then  accepted  as 
settled.  Koch  isolated  the  bacilli  in  pure  culture  and  demonstrated 
their  abilit}^  to  form  spores.  He  claimed  that  natural  infection  takes 
place  through  the  intestinal  tract,  although  he  was  at  first  unable 
to  demonstrate  this  by  feeding  food  infected  by  artificial  cultures. 
Later,  Pasteur  succeeded  in  producing  anthrax  by  feeding  hay 
sprayed  with  cultures  of  Bacillus  anthracis  when  he  at  the  same  time 
mixed  with  the  hay  thistles  or  any  plants  that  would  cause  pricking 
or  abrasions  of  the  intestinal  tract  so  that  the  bacilli  could  pass  into 
the  tissues. 

THE  ANTHRAX  BACILLUS. 

On  account  of  the  comparatively  large  size  of  the  Baeillus  anthra- 
cis— it  being  from  5  to  20  micromillimeters  long  and  from  1  to  1.5 
micromillimeters  broad,  although  both  longer  and  shorter  forms 
occur — and  the  great  readiness  and  certainty  with  which  it  kills 
experimental  animals,  it  was  the  earliest  disease  to  be  studied  with 
the  microscope  and  by  modern  bacteriological  methods.  We  first 
learned  to  demonstrate  spore  formation  and  spore  staining  from 
anthrax  cultures.  The  germ  is  exceedingly  easy  to  cultivate,  grow- 
ing on  almost  any  medium  a^d  at  a  Avide  range  of  temperature.  It 
has  many  of  the  general  morphological  characters  of  the  whole 
group  of  bacteria,  and  is,  therefore,  very  useful  for  teaching  pur- 
poses. The  bacillus  has  a  place  in  the  earliest  researches  on  im- 
munity, Pasteur  having  shown  in  1880-1882  that  animals  inoculated 
with  strains  of  the  bacilli  which  had  been  devitalized  to  a  certain 
extent  by  cultivation  at  high  temperatures  did  not  contract  anthrax 
when  exposed,  while  those  not  so  protected  did  contract  the  disease. 
Spores  of  anthrax  are  among  the  most  resistant  things  in  nature, 
and  are,  therefore,  useful  in  testing  the  germicidal  power  of  disin- 
fectants. 

If  a  drop  of  blood  is  taken  immediately  after  death  from  an  animal 
dead  from  anthrax  and  examined  with  a  microscope,  large  numbers 


THE  ANTHRAX  BACILLUS.  7 

of  nonmotile  bacteria  will  be  seen.  A  much  better  view  of  them 
can  be  obtained  if  a  so-called  blood-smear  preparation  is  stained 
with  methylene  blue  or  any  of  the  basic  anilin  dyes.  It  will  be 
noticed  that  the  ends  of  all  the  bacilli  are  sharply  cut  across.  If 
viewed  with  a  higher  power,  it  will  be  seen  that  the  ends  bulge  at 
the  corners,  making  the  ends  appear  slightly  concave.  The  cells  are 
also  noticed  to  have  a  capsule,  and  when  several  cells  are  lying  end 
to  end  the  capsule  seems  common  to  the  entire  chain  of  cells. 

If  in  making  preparations  of  anthrax  blood  great  care  is  taken 
not  to  overheat,  but  just  enough  to  "  set "  the  specimen,  and  methy- 
lene blue  is  used  as  the  stain,  we  can,  while  viewing  the  slide  by 
reflected  light  with  the  unaided  eye,  notice  a  violet  coloration  of  the 
entire  specimen.  If  w^e  now  place  the  slide  under  the  microscope, 
we  can  see  that  the  space  between  the  bacillus  and  its  capsule  is 
stained  a  reddish  color.  This  is  known  as  McFadyeans  color  test, 
and  if  the  specimen  is  properly  prepared  is  to  be  observed  in  all 
anthrax  preparations  made  from  dead  animals.  The  test  is  most 
successful  in  those  preparations  where  the  bacilli  are  very  numerous, 
and  could  be  of  great  importance  in  field  work  where  a  microscope 
may  not  be  at  hand. 

CULTURAL  AND  MORPHOLOGICAL  CHARACTERS. 

The  bacillus  of  anthrax  has  such  definite  and  constant  cultural  and 
morphological  characters  that  it  can  hardly  be  mistaken  for  any 
other  bacterium.  AVlien  gi'owing  on  the  surface  of  agar-agar  plate 
cultures  beautiful  colonies  occur,  appearing  as  wavy  wreaths  resem- 
bling locks  of  hair  or  bits  of  raw  cotton  thrown  on  the  surface.  It 
is  supposed  that  the  whole  mass  consists  of  thousands  of  contiguous 
cells  forming  one  long  chain,  wound  up  in  all  directions. 

The  growth  in  gelatin  tubes  is  characteristic  in  that  it  liquefies  the 
medium  and  the  growth  resembles  an  inverted  fir  tree. 

In  milk  a  small  amount  of  acid  is  formed,  so  that  milk  is  slightly 
coagulated  and  slowly  peptonized. 

In  peptone  bouillon  virulent  bacilli  make  a  much  more  luxuriant 
growth  than  do  the  attenuated  bacilli  or  vaccines.  The  heaviest 
growth  is  near  the  surface,  where  it  can  obtain  oxygen,  and  consists 
of  a  dense  mass  of  intertwined  bacilli  in  chains  which  readily  fall 
to  the  bottom  of  the  tube  if  it  be  even  slightly  shaken.  This  heavy 
growth  does  not  occur  when  bouillon  is  inoculated  with  attenuated 
bacilli. 

Growth  on  all  media  proceeds  best  at  35°  C.  The  minimal  tempera- 
ture for  growth  is  12°  C.,  and  the  maximal  45°  C.  The  vegetating 
bacillus  is  killed  by  a  short  exposure  (15  minutes)  to  60°  C.  moist 
heat,  and  by  desiccation  in  a  few  days  at  ordinary  temperatures.  The 
spores,  on  the  other  hand,  will  stand  almost  any  disinfecting  process 


8  ANTHRAX. 

that  any  infected  material  can  stand  without  greatly  changing  its 
physical  characters.  Hence  it  is  impossible  to  disinfect  hides  con- 
taining anthrax  spores  without  spoiling  them  for  leather  making. 
Spores  require  a  boiling  temperature  in  moist  heat,  and  a  much  higher 
temperature  in  dry  heat  to  destroy  them.  Spores  live  for  a  long 
time — probably  10  years — in  the  soil  or  in  water,  and  it  is  because 
of  this  great  resistance  that  pastures  once  infected  remain  so  for  an 
indefinite  period. 

The  bacillus  can  grow  either  in  the  presence  or  absence  of  oxygen, 
but  it  grows  best  when  air  or  oxygen  is  present.  Spores  can  form 
only  in  the  presence  of  oxygen,  and  this  accounts  for  the  fact  that 
the  bacillus  never  sporulates  in  the  living  blood  nor  in  cultures  grown 
under  anaerobic  conditions. 

When  spores  begin  to  form,  a  tiny  bright  spot  appears  in  the  center 
of  the  parent  cell,  which  soon  increases  to  the  diameter  of  the  cell 
and  always  remains  in  its  center.  The  cell  protoplasm  seems  to  be 
used  up  in  the  production  of  the  spore,  which  now  forms  the  resting 
stage  of  the  organism.  This  spore  remains  quiescent  until  it  is  again 
placed  in  a  suitable  environment  for  growth,  when  it  sprouts  and 
assumes  the  bacillary  or  infective  stage. 

Although  the  spore,  or  resting  stage,  of  Bacillus  anthraeis  is  very 
resistant  to  all  natural  and  artificial  disinfectants,  the  bacillar  or 
infective  stage  is  quite  vulnerable.  The  bacilli  locked  up  in  the  air- 
less blood  vessels  or  cavities  of  the  body  not  only  do  not  spore,  but 
rapidly  disintegrate  after  death.  These  important  facts  are  of  incal- 
culable value  in  the  suppression  of  the  disease  by  nature,  and  furnish 
the  strongest  kind  of  an  argument  for  the  prompt  burial  of  animals 
dead  from  the  disease.  If  such  an  animal  be  opened,  however,  the 
above-mentioned  natural  protection  against  the  spread  of  the  infec- 
tion is  removed,  as  the  bacilli  will  continue  to  multiply  and  sporula- 
tion  will  finally  ensue. 

The  following  experiments  were  made  to  demonstrate  the  foregoing 
facts:  The  spleen  of  a  guinea  pig  just  dead  from  anthrax  was  minced 
and  placed  in  a  Petri  dish  exposed  to  air  at  room  temperature. 
Microscopic  examinations  of  this  material  16  hours  after  showed  that 
every  bacillus  had  spored,  the  protoplasmic  cellular  material  staining 
poorly.  Another  experiment  made  to  determine  the  effect  of  such 
exclusion  of  the  air  as  burial  can  afford  showed  that  24  hours  are 
required  for  spore  formation. 

In  order  to  determine  the  effect  of  total  exclusion  of  air  upon  the 
question  of  viability  of  the  bacilli,  capillary  glass  tubes  were  filled 
with  heart's  blood  from  a  mouse  just  dead  from  anthrax  and  sealed 
in  a  flame.  These  sterile  tubes  were  filled  under  precautions  which 
prevented  the  ingress  of  extraneous  bacteria;  hence  there  were  only 
anthrax  bacteria  in  them,  and  the  disintegrating  effects  of  post- 


CHANNELS   OF  INFECTION.  9 

mortem  bacilli  through  the  septic  ferments  which  they  form  in  a 
decomposing  carcass  were  not  present.  Examinations  and  cultures 
from  these  tubes  were  made  at  intervals  up  to  the  twenty-third  day, 
and  while  the  bacilli  were  lessened  in  numbers,  they  were  still  viable. 
In  buried  carcasses,  even  though  they  be  not  opened,  putrefactive 
bactefia  grow  very  rapidly  and  their  excretions  cause  a  granular 
degeneration  of  the  anthrax  bacilli.  If  air  is  admitted  to  the  carcass 
these  excretions  have  no  deleterious  effect  upon  the  spores  that  have 
formed,  and  such  a  carcass  infects  the  soil  in  which  it  is  buried, 
while  if  the  carcass  had  not  been  opened,  and  care  had  been  taken 
to  disinfect  the  soiled  exterior  and  air-containing  cavities  of  the 
animal,  the  putrefactive  bacteria  would  kill  off  the  anthrax  bacilli. 
The  length  of  time  necessary  for  this  destruction  varies  with  the 
temperature,  being  about  a  week  in  hot  weather. 

CHAISTNELS  OF  INFECTION. 

There  are  four  routes  by  which  animals  may  become  infected, 
namely,  by  ingestion,  by  inoculation,  by-  inhalation,  and  by  trans- 
mission. 

Ingestion. — The  first  and  most  frequent,  and  therefore  the  most 
important,  is  by  ingestion.  This  form  is  acquired  when  animals  are 
turned  out  to  pasture. 

Inoculation. — The  method  of  infection  next  in  importance  is  by 
inoculation,  which  is  brought  about  by  contact  of  abrasions  and 
wounds  with  infected  material,  or  by  flies  or  other  insects. 

A  case  of  spontaneous  inoculation  arising  in  the  course  of  my  ex- 
periments— the  only  one  which  occurred  in  the  work — was  where  a 
ram,  immune  to  anthrax,  was  severely  bled  to  furnish  blood  for 
experiments.  He  was  kept  in  a  stall  where  a  sheep  had  died  three 
months  previously  of  experimental  anthrax.  A  considerable  wound 
was  made  by  the  operation  and  the  animal  was  found  dead  of  an- 
thrax on  the  second  day  after  having  been  bled. 

I  Inhalation. — The  form  third  in  importance  is  by  inhalation.  Here 
spores  are  breathed  in  with  dust  arising  from  infected  material  or 
surfaces. 

Transmission. — A  fourth  form,  which  is  probably  rare,  is  that  ot 
transmission  from  the  mother  to  the  fetus  in  utero.'  I  have  observed 
this  in  the  case  of  an  experimental  cow  which  had  been  immunized 
with  a  single  anthrax  vaccine  and  subsequently  tested  with  virulent 
bacilli.  The  mother  at  no  time  appeared  sick,  but  on  the  sixteenth 
day  after  receiving  the  virulent  bacilli  she  dropped  a  dead  calf, 
which  appeared  to  be  a  full-term  one.  Not  suspecting  the  cause  of 
the  trouble,  the  calf  and  membranes  were  buried.  Fortunately,  a 
loop  of  the  discharge  found  on  the  ground  was  plated  and  develojjed 
a  pure  cultivation  of  60,000  anthrax  bacilli,  the  virulence  of  which 
89049°— Bull.  137—11 2 


10  ANTHRAX. 

was  demonstrated  on  a  guinea  pig.  The  cow  was  in  no  way  sick, 
and  daily  microscopic  examinations  of  the  vaginal  discharges  showed 
these  were  free  of  anthrax  bacilli. 

INFECTION  OE  THE  SOIL. 

Soil  becomes  infected  with  anthrax  by  discharges  from  animals 
suffering  with  the  disease,  and  by  the  dead  bodies  of  animals  which 
are  allowed  to  decompose  upon  the  surface.  These  discharges  con- 
sist of  small  hemorrhages  which  occur  from  the  mucous  membranes 
in  the  nose  and  bowels,  mixed  with  the  nasal  secretions  and  with 
the  feces.  These  sometimes  do  not  occur  until  just  before  death. 
In  some  cases  the  discharges  are  so  limited  as  almost  to  escape  obser- 
vation, but  a  microscopic  examination  in  such  cases  would  reveal 
large  numbers  of  bacilli.  Hence  it  is  very  important  to  limit  to  the 
smallest  area  possible  the  wanderings  of  an  animal  so  affected.  It  is 
doubtful  if  the  feces  of  the  smaller  experimental  animals  contain 
anthrax  bacilli.  One  observer  reports  the  presence  of  anthrax  spores 
in  the  feces  of  guinea  pigs,  but  I  have  never  found  them. 

Pasteur,  in  his  memorable  paper  on  anthrax  before  the  Academy 
of  Sciences  in  Paris  in  1880,  gave  his  opinion  as  to  another  manner  in 
which  the  surface  becomes  infected  with  this  disease.  He  said 
in  part ; 

Earthworms  are  the  bearers  of  the  germs,  and  they  bring  this  terrible  para- 
site to  the  surface  of  the  ground  from  the  depths  to  which  it  has  been  buried. 
It  is  in  the  little  cylinders  of  earth,  of  fine  earthy  particles,  that  the  worms  ex- 
crete and  deix)sit  at  the  surface  of  the  ground  after  morning  dews,  or  after 
rains,  that  are  to  be  found,  besides  a  crowd  of  other  germs,  the  germs  of 
charbon  (anthrax). 

While  it  does  not  seem  impossible  that  earthworms  may  swallow 
the  spores  of  anthrax  and  cast  them  out  upon  the  surface,  that  close 
observer,  Koch,  was  unable  to  verify  the  facts.  He  held  that  pas- 
tures remain  infected  on  the  surface  from  preexisting  cases,  and 
that  the  parasite  remains  on  or  near  the  surface  from  season  to 
season  and  infects  animals  which  graze  upon  such  soil.  When  the 
grass  gets  short  in  dry  weather,  spores  that  may  have  been  carried 
into  the  soil  by  previous  rains  either  rise  again  by  capillarity  or  are 
brought  to  the  surface  by  the  animals  pulling  the  grass  up  by  the 
roots.  The  writer  has  examined  many  earthworm  casts  from  an- 
thrax graves  of  various  ages — from  15  years  to  1  month — and  has 
failed  to  find  anthrax  spores  in  them.  Pasteur's  work  was  done  be- 
fore the  days  of  solid  media,  and  hence  the  plate-culture  method  of 
isolation  was  not  used  by  him.  Another  disease,  malignant  edema, 
is  easily  produced  in  guinea  pigs  and  rabbits,  and  greatly  resembles 
anthrax  clinically,  as  well  as  in  the  morphology  of  the  organism 


METHODS  OF   DIAGNOSIS.  11 

causing  it.    These  two  germs  would  greatly  resemble  each  other 
when  viewed  with  a  microscope  made  in  1880. 

In  order  to  test  further  the  question  as  to  whether  the  surface  above 
an  anthrax  grave  becomes  infected  by  earthworms,  or  otherwise,  the 
writer  has  for  the  past  three  years  pastured  sheep  and  cattle  upon 
soil  in  which  there  are  many  anthrax  graves,  and  no  case  of  spontane- 
ous anthrax  has  arisen.  The  animals  were  frequently  observed  graz- 
ing in  the  little  hollows  formed  by  the  sinking  in  of  the  graves,  and 
it  would  seem  that  this  is  sufficient  proof  of  the  questionableness  of 
Pasteur's  early  theory  regarding  this  source  of  infection. 

METHODS  OF  DIAGNOSIS. 

Anthrax  is  a  disease  that  we  absolutely  know  is  caused  by  the  para- 
site ascribed  to  it.  We  can  tell  just  how  long  an  animal  will  live  after 
it  has  been  inoculated  with  a  given  culture,  provided  the  culture  is  of 
maximum  strength.  We  know  that  a  virulent  culture  will,  in  proper 
dose  kill  the  laboratory  animals,  as  well  as  sheep,  in  48  hours,  and 
that  we  can  always  recover  the  germ  in  the  blood  of  such  animals 
and  reproduce  the  same  disease  in  another  animal  by  inoculating  it 
with  this  blood.    Hence,  all  of  Koch's  postulates  can  be  fulfilled. 

Different  races  of  anthrax  bacilli  vary  in  virulence.  Those  of 
highest  virulence  will  kill  guinea  pigs  and  rabbits  in  0.00001  c.  c. 
doses  of  a  24-hour  bouillon  culture  in  48  hours,  and  the  same  culture 
would  kill  a  sheep  in  95  hours  when  given  a  0.0001  c.  c.  dose  subcu- 
taneously.  Very  young  animals  are  much  more  susceptible  to  anthrax 
than  adults,  and  hence  care  must  be  exercised  in  vaccinating  young 
animals.  A  sucking  rabbit  a  few  days  old  died  in  18  hours  from  a 
0.1  c.  c.  dose  of  bouillon  culture,  and  the  virulence  of  the  bacillus, 
which  had  become  somewhat  lessened,  was  gi'eatly  enhanced. 

A  diagnosis  can  be  made  by  the  veterinary  scientist  by  two  meth- 
ods; either  by  a  bacterioscopic  examination  of  blood  smears  made  soon 
after  death  or  by  animal  inocuhitions.  On  account  of  the  nature  of 
the  disease  it  is  highly  desirable  that  the  first  method  be  used,  as  no 
time  should  be  lost  in  coming  to  a  decision.  It  is  easy  to  reach  a  de- 
cision if  the  specimen  has  been  properly  prepared  and  if  the  micro- 
scopist  is  familiar  with  the  organism. 

PREPARATION    OP   SPECIMENS. 

A  small  drop  of  blood  thinly  spread  on  a  piece  of  glass  or  paper 
and  dried  immediately  is  all  that  is  required.  It  is  best  to  obtain 
blood  for  this  purpose  by  making  a  needle  puncture  into  the  jugular 
vein,  as  sometimes  the  bacilli  are  not  numerous  in  the  peripheral 
blood  vessels.  A  specimen  prepared  in  this  way  will  arrive  at  the 
laboratory  just  in  the  condition  in  wliicli  it  was  collected.  There 
will  be  no  bacteria  in  it  that  were  not  there  when  it  was  prepared. 


12  ANTHRAX. 

Sometimes  specimens  arrive  consisting  of  two  smeared  surfaces  of 
glass  stuck  together.  These  are  dangerous,  and  it  is  next  to  impos- 
sible to  separate  them.  They  should  be  boiled  and  thrown  away,  no 
attempt  being  made  to  make  an  examination. 

The  most  dangerous  anthrax  package,  and  one  which  is  not  allowed 
in  the  mails,  is  the  bottle  of  blood.  These  have  arrived  broken,  and 
in  one  instance  a  positive  diagnosis  was  made  from  blood  smeared  on 
the  outside  of  the  package  by  the  sender's  hands.  Again,  when  parts 
of  organs,  or  blood  in  volume,  are  sent  in,  decomposition  will  have 
taken  place  and  a  diagnosis  may  be  impossible.  The  fruit- jar  pack- 
age, containing  spleen,  liver,  heart,  etc.,  sent  by  express,  is  another 
most  dangerous  package.  These  have  arrived  broken,  with  liquid 
dripping  from  them.  When  not  broken  their  opening  is  generally 
attended  with  dangerous  difficulties.  The  top  is  usually  screwed 
down  so  tight  that  considerable  force  must  be  used  to  open  it,  and 
when  this  is  accomplished  the  gases  which  have  formed  will  spray 
the  liquids  upon  one's  hands  and  clothing. 

A  diagnosis  of  a  blood-smear  preparation  is  made  by  first  "  set- 
ting" the  specimen  by  heat,  passing  the  glass  slide  through  the  flame 
three  times,  occup^nng  a  second  each  time.  The  specimen  is  now 
stained  with  methylene  blue  or  fuchsin,  dried  between  two  pieces  of 
filter  paper,  and  mounted  for  examination.  If  anthrax  bacilli  are 
present  they  can  be  easily  seen  stained  blue  or  red,  according  to  the 
stain  used.  They  appear  as  rods  with  square  ends.  Tliey  are  never 
round  or  blunt.  They  will  almost  always  appear  in  chains,  although 
they  frequently  appear  as  single  cells  when  torn  apart  in  making  the 
preparation.  Spores  are  never  found  in  a  specimen  prepared  from  a 
recently  dead  animal.  With  lenses  of  1,000-1,200  amplification  the 
ends  appear  somewhat  concave,  from  bulging  of  the  corners  of  the 
cylinder.  Wlien  such  an  organism  is  found  in  the  blood  of  an  animal 
recently  dead  the  disease  may  be  called  anthrax,  as  it  is  the  only  one 
in  animals  that  provides  such  a  picture. 

ANIMAL.  INOCULATIONS. 

When  there  is  reason  for  doubt,  animal  inoculation  must  be  re- 
sorted to.  Mice,  guinea  pigs,  and  rabbits  may  be  used,  and  these  are 
susceptible  in  the  order  named.  The  suspected  material  is  ground 
up  in  a  sterile  mortar  containing  normal  salt  solution.  The  solid 
particles  are  allowed  to  settle  or  are  filtered  off,  and  a  few  drops  to 
0.5  c.  c.  of  the  liquid  are  injected  subcutaneously  into  guinea  pigs. 
If  anthrax  be  present  the  animal  will  die  in  two  or  three  days,  and 
the  bacillus  can  be  recovered  from  its  blood  and  examined  as  in  the 
first  method.     Plate  cultures  should  also  be  made  from  the  extract 


DIFFERENT  FORMS   OF   ANTHRAX.  13 

of  the  suspected  substance,  as  it  is  possible  to  isolate  the  anthrax 
organism  and  thus  come  to  a  decision  from  a  study  of  the  anthrax 
colonies  if  any  develop,  as  such  colonies  have  a  characteristic  appear- 
ance. Inoculation  into  experimental  animals  of  cultures  made  from 
these  colonies,  if  they  be  anthrax,  will  cause  the  disease,  and  thus  any 
doubt  as  to  their  identity  will  be  removed. 

DIFFERENT   FORMS   OF   ANTHRAX. 

Anthrax  manifests  itself  in  different  forms  according  to  the  seat  of 
invasion,  kind  of  animal,  virulence  of  the  bacillus,  season  of  the  year, 
and  time  of  the  outbreak. 

The  most  acute  type  of  the  disease  is  seen  oftenest  in  cattle  and 
sheep  and  is  known  as  apoplectic  or  fulminant  anthrax.  This  form 
appears  suddenly,  without  premonitory  symptoms.  The  animal  is 
suddenly  seized  with  trembling,  a  haggard  expression,  swaying,  diffi- 
cult breathing,  and  cyanosis.  Convulsions  and  death  occur  in  from 
a  few  minutes  to  two  or  three  hours.  Blood}'  discharges  usually 
occur  during  the  last  hour  from  the  nose  and  rectum.  These  are 
very  infectious,  as  the  bacilli  are  present  in  large  numbers,  and 
constitute  an  important  way  in  which  pastures  become  infected. 
For  this  reason  such  animals  should  be  confined  where  these  dis- 
charges can  be  .disinfected. 

The  second  form,  known  as  anthrax  fever  or  internal  anthrax, 
differs  from  the  fulminant  or  apoplectic  form  only  in  its  duration. 
The  symptoms  in  the  last  stages  are  practically  those  seen  in  the 
most  acute  type,  except  that  they  are  more  intense  and  of  longer 
duration.  There  will  be  excitability,  restlessness,  high  fever,  oozing 
of  blood  from  the  nose,  eyes,  ears,  rectum,  and  thinner  parts  of 
the  skin  of  the  axilla  or  thigh ;  tremors,  dullness,  prostration,  grind- 
ing of  the  teeth,  colicky  pains,  difficult  breathing,  arching  of  the 
back,  rolling  of  the  eyes,  convulsions,  and  death  in  sheep  in  24 
hours,  in  -^attle  in  from  2  to  5  days,  and  in  horses  in  from  1  to  5 
days. 

The  third  form  is  local  or  external  anthrax.  In  this  form  the 
disease  is  at  first  localized  in  the  mouth,  throat,  or  skin  in  cattle, 
and  the  same  form  appears  most  often  in  horses  in  the  tongue, 
throat,  neck,  breast,  withers,  shoulders,  flank,  or  thigh.  These 
swellings  are  firm  or  doughy,  insensitive  at  certain  parts,  and  tend 
to  become  gangrenous.  There  is  no  tendency  to  suppuration,  and 
they  do  not  crackle  on  pressure — a  diagnostic  point  when  there  is 
doubt  as  to  whether  the  case  is  one  of  anthrax  or  of  blackleg.  When 
cut  into — something  which  should  never  be  done,  as  the  exudation 
always  contains  the  anthrax  bacillus — there  escapes  either  a  pale, 
straw-colored,  or  bloody  liquid. 


14  ANTHRAX. 

SYMPTOMS. 

The  sj^mptoms  of  anthrax  will  vary  with  the  species  and  with  the 
type  of  the  disease,  except  that  in  the  last  stages  of  any  of  the  three 
types  the  most  pronounced  symptoms  are  identical.  In  the  most 
acute  type  the  animal  may  appear  at  first  to  be  perfectly  well  and 
keep  along  with  its  fellows  even  when  its  temperature  is  very  high — 
as  high  as  106°  F.  Along  with  such  a  temperature  we  shall  of  course 
find  a  rapid  pulse  and  increased  respiration.  When  one  is  standing 
close  beside  such  an  animal  the  heart  beats  may  be  plainlj'^  heard. 
Soon  other  symptoms,  such  as  grinding  of  the  teeth,  tremors,  and 
standing  with  head  down,  appear.  Then  appear  drooping  of  the 
head  and  ears  and-. a  disposition  to  lie  down.  Animals  that  have  been 
lively  will  now  decline  to  rise  unless- handled  roughly.  Thej'^  become 
stupid  and  sleepy  and  very  weak  in  the  hind  parts.  Whereas  the 
temperature  has  all  along  been  high,  it  now  shows  a  sharp  decline, 
and  before  death  may  become  subnormal.  The  visible  mucous  mem- 
branes are  a  dusky  red,  especially  those  of  the  rectum  and  vulva. 
There  is  a  bloody  nasal  discharge.  The  feces  will  be  coated  with  a 
bloody  mucus.  Local  swellings  appear  in  the  mouth,  throat,  neck, 
and  breast  (especially  in  horses),  and  there  are  sharp  attacks  of  colic 
and  convulsions  which  end  the  misery  of  the  animal  in  from  12  to  48 
hours  after  the  disease  is  first  noticed.  Pregnant  annuals  are  liable 
to  abort  and  thus  greatly  spread  the  infection  through  the  copious 
discharges.  An  outbreak  has  its  highest  mortality  at  its  onset,  while 
later  on  some  animals,  especially  horses  and  mules,  may  recover.  In 
the  most  acute  types,  which  occur  mostly  in  cattle  and  sheep,  the 
animal  is  found  dead.  A  cow  which  seemed  well  at  night  is  found 
dead  in  the  morning,  or  if  death  occurs  in  the  d*aytime,  the  illness  is 
of  short  duration,  occupying  only  a  few  minutes  or' one  or  two  hours. 
In  these  sudden  attacks  the  symptoms  follow  each  other  so  rapidly 
and  death  is  so  sudden  that  sometimes  the  owner  is  convinced  that 
the  animal  has  been  poisoned.  The  attack  is  ushered  in  with  trem- 
bling, anxious  expression,  high  fever,  rolling  of  the  eyes,  and  con- 
vulsive movements,  soon  followed  by  general  convulsions  and  death. 

In  the  local  form  or  cutaneous  anthrax  in  cattle,  swellings  appear 
suddenly  on  different  parts  of  the  body  at  one  or  many  places,  and 
the  animal  dies  with  the  same  symptoms  as  occur  in  the  most  acute 
type  when  the  bacilli  reach  the  circulating  blood  from  these  local 
lesions.  When  the  infection  occurs  in  the  tongue  or  pharnyx,  we  have 
in  the  first  case  gloss  anthrax,  and  in  the  latter  pharyngeal  anthrax, 
the  ^mptoms  varying  somewhat  according  to  the  part  most  affected ; 
but  the  general  constitutional  symptoms  will  be  those  already  de- 
scribed. In  some  cases  the  most  prominent  symptoms  at  first  will 
be  enormous  swellings  of  the  rectal  mucous  membrane. 


POST-MOETEM  APPEARANCES.  15 

In  the  local  form,  or  cutaneous  anthrax,  in  the  horse,  the  swellings 
occur  at  the  points  of  entrance  of  the  bacilli  or  spores,  where  there 
are  abrasions  of  the  skin  or  mucous  membrane,  or  where  biting  in- 
sects have  brought  the  infection  from  a  previous  case.  These  swell- 
ings appear  suddenly  at  the  point  of  inoculation  and  are  character- 
ized by  a  rapidly  spreading  edema.  The  general  symptoms  are  not 
so  urgent,  the  fever  is  less  intense,  and  the  mortality,  while  not  so 
great  as  in  the  more  acute  form,  is  still  high. 

POST-MORTEM  APPEARANCES. 

An  animal  that  has  died  of  anthrax  will  nearly  always  be  found 
much  bloated,  with  blood  oozing  from  the  nose  and  rectum.  There 
will  be  evidences  on  the  ground  that  the  animal  died  a  violent  death, 
in  convulsions.  Local  swellings  will  be  present  or  absent  according 
to  the  type  of  the  disease.  In  the  rapidly  fatal  cases  little  change 
will  be  noted  either  in  the  blood  or  internal  organs  beyond  those 
produced  by  high  blood  pressure,  indicated  by  a  swollen  spleen  and 
engorged  liver.  The  carcass  itself — the  edible  portions — would 
show  nothing  that  would  make  it  doubtful  as  food.  In  the  more 
prolonged  cases  a  hemorrhagic  condition  will  be  noted  in  all  the 
internal  organs.  The  blood  will  be  tarry  in  appearance  and  will 
not  clot.  The  heart  is  often  light  in  color,  while  on  the  inside  it 
will  be  found  deeply  stained  and  containing  dark,  uncoagulated 
blood.  The  liver  may  be  found  enlarged  and  is  easily  torn  in  han- 
dling, presenting  on  its  surface  hemorrhagic  areas.  The  spleen  is 
often  specially  enlarged  and  distorted  in  shape,  and  ruptures  on 
handling. 

The  bacilli  of  anthrax  can  be  found  in  largest  numbers  in  those 
organs  where  the  lesions  are  most  pronounced,  namely,  the  spleen, 
liver,  and  engorged  lymphatic  glands,  but  they  are  commonly  found 
in  any  part  of  the  vascular  system.  In  the  serous  cavities,  such  as 
the  pericardial,  the  pleural,  and  the  peritoneal,  may  be  found  a  san- 
guineous fluid  consisting  of  serum,  red  and  white  cells,  and  anthrax 
bacilli.  The  hyperemia  and  areas  of  extravasated  blood  may  appear 
at  any  point  of  the  body  where  the  bacilli  have  become  localized  and 
enormously  multiplied,  forming  capillary  embolisms,  which  consist 
of  broken-down  blood  corpuscles,  and  bacilli.  Hence  they  are  fre- 
quent in  the  tongue,  throat,  lungs,  stomach,  and  intestinal  walls;  the 
mesentery  and  the  omentum;  the  skin,  connective  tissue,  and  the 
muscles. 

THE    CAUSE   or   DEATH. 

From  the  manner  of  death  it  would  seem  that  the  poison  of  an- 
thrax acts  specifically  upon  the  center  of  respiration,  this  in  turn 
allowing  a  fatal  accumulation  of  carbon  dioxid  in  the  blood.    That 


16  ANTHRAX. 

it  is  the  replacement  of  the  oxygen  of  the  blood  by  carbon  dioxid  that 
is  the  immediate  cause  of  death  in  antlirax  is  further  shown  by  the 
loss  in  the  blood  of  the  property  of  coagulation,  it  being  known  that 
carbon  dioxid  precipitates  and  throws  out  of  action  the  fibrin-form- 
ing element  of  the  blood — fibrinogen. 

DISPOSAL  OF  CAECASSES. 

The  disposal  of  an  anthrax  carcass  should  not  be  left  to  the  owner 
nor  should  anyone  be  allowed  to  open  a  carcass  of  an  animal  that  is 
supposed  to  have  died  from  anthrax.  The  diagnosis  can  only  with 
certainty  be  made  by  a  competent  bacteriologist.  He  need  only  be 
furnished  a  drop  of  blood  from  the  suspected  animal.  This  blood 
can  be  drawn  from  the  jugular  by  means  of  a  syringe,  or  a  small  nick 
sulficient  to  allow  a  drop  to  escape  may  be  made  with  a  suitable  in- 
strument. This  blood  should  be  smeared  upon  a  piece  of  glass  or 
piece  of  paper,  dried  immediately,  and  forwarded  to  the  bacteriolo- 
gist. Any  blood  escaping  during  the  above  operation  should  at  once 
be  disinfected.  The  State  should  appoint  a  sanitary  agent  in  every 
election  district,  whose  duty  it  should  be  to  dispose  of  anthrax  car- 
casses, according  to  instructions  issued  by  a  competent  authority,  as 
it  is  only  by  the  proper  disposal  of  the  carcass  and  by  vaccination  that 
the  disease  can  be  controlled.  While  the  majority  of  our  farmers 
recognize  the  advisability  of  promptly  burying  the  carcass,  there  are 
those  who  would  allow  the  animals  to  lie  where  they  die  to  decompose 
or  be  eaten  by  dogs  and  buzzards.  Parts  of  the  carcass  may  be  car- 
ried bj'^  dogs  to  adjoining  or  distant  pastures  and  spread  the  infection. 
Spores  forming  in  the  carcass  opened  in  this  way  would  pass  through 
the  intestinal  canal  of  buzzards  and  dogs  and  permanently  infect 
pastures  miles  away.  The  soil  upon  which  such  an  animal  has  lain 
would  certainly  be  infected  and  remain  so  for  many  years. 

During  the  last  few  years  deep  burial,  rather  than  efforts  at  crema- 
tion, has  been  practiced,  with  good  results  so  far  as  can  be  ascertained. 
While  cremation  properly  carried  out  is  undoubted^  the  safest 
method  of  disposing  of  an  anthrax  carcass,  it  is  practically  impos- 
sible to  destroy  absolutely  all  vestiges  of  the  body  and  bacilli-laden 
bod}'  fluids,  and  unless  this  is  done  the  whole  operation  so  far  as  it 
goes  is  almost  a  useless  waste  of  time,  material,  and  labor.  In  the 
cremation  of  carcasses  in  the  field  even  the  earth  upon  which  it  has 
lain  should  be  thoroughly  and  deeply  burned  over  so  that  the  heat 
will  penetrate  to  a  depth  sufficient  to  insure  the  death  of  the  anthrax 
bacilli  which  certainly  passed  into  the  soil  with  the  body  fluids  when 
rupture  occurred  from  the  heat.  We  know  enough  of  the  biology  of 
the  anthrax  bacillus  to  take  advantage  of  the  fact  that  if  it  is  kept 
locked  up  in  the  closed  cavities  and  blood  vessels  of  the  body  where 
no  air  can  reach  it,  not  only  will  the  bacilli  fail  to  produce  the  re- 


DISPOSAL.  OF   CARCASSES.  17 

sistant  spore,  but  they  will  actually  decompose  and  become  noninfec- 
tious, so  that  the  bacilli  which  are  in  the  blood  vessels  of  an  unopened 
animal  perish.  On  the  other  hand,  those  bacilli  which  have  come 
away  in  the  pulmonary  and  rectal  discharges  during  life  will,  if  they 
are  provided  with  sufficient  material  to  keep  them  moist,  promptly 
go  into  the  spore  or  resting  stage.  These  constitute  the  main  source 
of  danger,  and  in  this  way  a  considerable  area  may  become  infected. 
For  similar  reasons,  hemorrhages  into  atr-containing  cavities,  such  as 
the  lungs,  trachea,  head,  sheath,  vagina,  and  rectum,  all  favor  the 
production  of  the  resistant  spore.  Hence,  if  we  introduce  into  the 
cavities  a  disinfectant  which  will  surely  kill  the  spores,  and  also  at 
the  same  time  take  care  to  disinfect  the  soiled  exterior  surfaces  of 
the  animal,  and  then  bury  such  an  animal  without  opening  it,  we  may 
reasonably  conclude  that  such  a  carcass  will  not  thereafter  be  a 
source  of  infection. 

THE  OFFICIAL  METHOD  IN   DELAWARE. 

In  Delaware  the  method  of  procedure  as  advised  and  as  carried  out 
by  the  State  board  of  agriculture  is  as  follows :  Formalin  is  injected 
in  both  directions  into  the  trachea.  Formalin-soaked  cotton  is  pressed 
up  the  nostrils  and  into  the  mouth,  rectum,  vagina,  or  sheath.  The 
grave  is  dug  where  the  animal  dies,  when  possible.  The  body  of  the 
animal  is  then  either  thoroughly  wetted  down  with  a  4  per  cent  solu- 
tion of  formalin  or  kerosene,  or  coal  oil  is  poured  over  the  uppermost 
side  of  the  animal  and  is  set  on  fire.  Only  enough  kerosene  is  applied 
to  burn  off  the  hair,  as  it  would  defeat  the  purpose  to  burn  the  animal 
sufficiently  to  cause  rupture  of  the  body.  The  carcass  is  then  rolled 
into  the  grave  with  the  other  side  uppermost.  This  side  is  now 
sprinkled  with  the  oil  and  fired,  after  which  the  dirt  upon  which  the 
animal  has  lain  is  disinfected  with  formalin  and  is  then  shoveled  into 
the  grave  first.  The  grave  is  now  filled  and  banked  up  as  is  the  cus- 
tom. Such  graves  should  be  surrounded  by  a  fence.  In  fact,  there 
should  be  a  graveyard  widely  fenced  in,  and  when  an  animal  is  known 
to  have  anthrax,  it  should  be  placed  therein  and  allowed  to  die  where 
it  is  to  be  buried.  Such  an  inclosure  should  never  be  used  for  any 
other  purpose,  and  should  be  so  located  that  drainage  from  it  upon 
other  land  will  not  occur. 

SUSCEPTIBILITY  OF   THE   VARIOUS   ANIMALS. 

While  all  the  farm  animals  and  even  man  himself  will  contract 

anthrax,  we  know  that  the  degree  of  susceptibility  varies  not  only  in 

the  different  species,  but  also  in  individuals  of  the  same  species.     We 

find  that  the  herbivora  are  very  susceptible  and  that  the  carnivora 

89049°— Bull.  137—11 3 


18  ANTHEAX. 

are  less  so,  while  the  omnivora  occupy  a  place  between  these  two 
extremes.  Cattle  and  sheep,  being  extremely  susceptible,  contract 
the  fulminating  type  in  nearly  every  case,  while  horses  and  mules 
frequently  live  several  days,  exhibiting  the  local  or  skin  form,  and 
some  cases  recover.  It  could  probably  be  shown  that  any  ruminant 
is  more  susceptible  than  other  herbivora  because  of  the  anatomical 
and  physiological  characteristics  of  their  respective  digestive  organs. 
In  ruminants  we  find  the  immense  rumen,  with  its  moisture,  heat, 
and  alkaline  reaction,  an  admirable  incubator  for  bacteria.  The  out- 
ward passage  of  the  contents  of  the  rumen  is  slow,  so  there  is  plenty 
of  time  for  anthrax  spores  swallowed  with  the  food  to  develop  into 
bacilli  and  multiply  into  prodigious  numbers  before  they  pass  through 
the  other  two  stomachs  into  the  true  or  acid  stomach,  where  some  of 
them  may  be  digested,  but  where  many  may  pass  out  unharmed  into 
the  alkaline  intestine  and  be  absorbed  with  the  food,  finally  entering 
the  blood  stream  suspended  in  the  chyle.  It  would  now  require  only 
a  few  hours  for  them  to  multiply  in  the  blood  into  sufficient  numbers 
to  produce  their  fatal  poison.  It  is  doubtless  in  this  way  that  cattle 
which  seem  well  when  last  seen  and  fed  at  night  come  down  suddenly 
with  the  disease  and  are  found  dead  in  the  morning. 

Swine  are  less  susceptible  than  horses  and  cattle,  yet  when  swine 
eat  the  carcasses  of  animals  dead  from  anthrax  they  may  contract 
the  disease.  As  in  other  species,  the  young  sucking  pigs  are  more 
susceptible  than  the  adults.  As  swine  usually  contract  the  disease 
by  eating  the  dead  animals,  they  generally  have  the  disease  in  the 
throat  or  intestine.  The  symptoms  in  such  cases  do  not  vary 
materially  from  like  cases  in  the  other  species. 

Dogs  and  cats  contract  the  disease  similiarly  to  hogs  and  are 
classed  among  the  less  susceptible.  The  symptoms  are  those  to  be 
expected,  and  consist  of  swelling  of  the  throat,  difficult  swallowing, 
vomiting,  profuse  bloody  diarrhea,  high  fever,  and  death. 

Birds  of  prey  are  said  to  be  immune  to  the  disease.  Chickens  are 
less  susceptible  than  swine,  yet  Pasteur  showed  that  chickens  were 
quite  susceptible  when  immersed  in  cold  water  and  then  inoculated. 
Likewise  frogs,  which  are  ordinarily  immune,  can  be  successfully 
inoculated  after  immersion  in  warm  Avater.  Young  pigeons  of 
certain  breeds  are  easily  infected  artificially,  and  sparrows,  finches, 
canaries,  yellow-hammers,  and  redbreasts  have  been  successfully 
inoculated. 

In  chickens  anthrax  runs  a  very  rapid  and  fatal  course  in  24 
hours.  They  can  contract  the  disease  by  eating  the  carcass  or  dis- 
charge of  an  animal  dead  from  anthrax.  As  in  other  animals,  a 
differential  diagnosis  between  this  disease  and  other  chicken  diseases 
of  like  type  should  be  based  upon  the  discovery  of  the  anthrax 
bacillus  in  the  blood  along  with  the  symptoms  of  sudden  debility 


ANTHRAX  IN   MAN.  19 

and  high  fever.  Anthrax  swellings  will  occur  on  the  comb  and 
wattles,  around  the  eyes,  and  on  the  tongue,  palate,  and  feet.  The 
bird  is  extremely  weak,  has  violent  tremors  and  convulsions,  and 
bloody  diarrhea,  and  dies  within  24  hours. 

ANTHRAX  IN  MAN. 

Anthrax  is  not,  strictly  speaking,  a  human  disease,  yet  it  is  not 
rare  in  the  practice  of  physicians  who  attend  the  workers  in  tanneries 
where  imported  hides  are  converted  into  leather,  nor  is  it  rare  in  per- 
sons whose  occupation  brings  them  in  otherwise  contact  with  hides, 
wool,  hair,  furs,  hoofs,  bones,  rags,  felt,  glue,  or  any  product  made 
from  the  bodies  of  animals  that  die  of  anthrax.  Hence  the  disease  is 
found  in  shepherds,  cattlemen,  horsemen,  farmers,  drovers,  butchers, 
tanners,  brush  makers,  and  veterinarians. 

The  disease  is  notoriously  common  in  those  who  handle  imported 
hides,  although  man  can  become  infected  through  the  bites  of  flies  or 
other  infected  insects.  The  sound  skin  is  sufficient  protection  against 
the  entrance  of  the  bacillus,  but  the  slightest  abrasion  is  sufficient  as  a 
point  of  inoculation  in  handling  infected  material,  or  even  for  inocu- 
lation by  the  ordinary  housefly  that  has  visited  the  discharges  of  an 
animal  suffering  with  anthrax.  The  process  of  tanning  does  not 
always  disinfect  an  anthrax  hide.  That  hair  is  a  medium  of  infec- 
tion is  shown  by  outbreaks  of  anthrax  among  brush  makers.  Per- 
sons who  handle  imported  wool  and  rags  contract  the  disease  by  in- 
haling the  spores  in  the  dust  which  arises.  This  spore  develops  into 
the  bacillus,  and  a  case  of  pulmonary  anthrax  is  established.  This 
form  is  known  as  woolsorters'  disease,  or  ragpickers'  disease.  Those 
who  handle  bones  and  carcasses  in  fertilizer,  glue,  and  rendering 
establishments  located  in  anthrax  districts  are  peculiarly  exposed  to 
infection.  In  man  the  large  majority  of  cases  occur  on  the  face,  prob- 
ably because  this  part  of  the  body  is  more  liable  to  be  attacked  by 
biting  insects. 

Anthrax  in  man  is  a  local  disease,  and  we  do  not  find  the  bacilli  in 
as  large  numbers  in  the  blood  or  other  organs,  such  as  the  liver, 
spleen,  and  kidneys,  as  we  do  in  the  lower  animals.  The  bacilli  can, 
however,  be  found  in  abundance  in  the  local  lesion. 

MALIGNANT  PUSTULE. 

Malignant  pustule,  or  local  anthrax  of  the  skin,  usually  occurs  on 
the  face,  hands,  arms,  or  neck  of  those  who  handle  imported  hides. 
In  from  one  to  three  days  after  infection  takes  place  a  small  red 
pimple  appears.  This  soon  changes  to  a  vesicle,  which  is  very  pain- 
ful. The  center  of  the  vesicle  rapidly  becomes  necrotic,  fonning  a 
black  eschar,  which  soon  becomes  surrounded  by  a  ring  of  vesicles. 


20  ANTHRAX. 

The  surrounding  tissues  become  congested  and  edematous,  and  the 
lymphatics  are  involved.  The  fever  is  quite  high,  and  the  patient 
is  very  sick  from  the  absorption  of  poison  from  the  local  disease.  It 
runs  a  rapid  course,  and  the  patient  may  die  from  toxemia. 

Most  of  these  cases  are,  fortunately,  susceptible  of  successful  treat- 
ment. This  consists  of  early  and  thorough  excision  of  the  pustule 
and  all  infected  surrounding  tissue,  followed  by  the  local  application 
of  strong  disinfectants.  At  Guy's  Hospital,  in  London,  13  out  of  15 
cases  were  cured  by  excision,  even  though  in  12  of  these  cases  the 
inflammation  had  spread  to  the  surrounding  parts  or  had  involved 
the  lymphatic  glands  with  more  or  less  severe  constitutional  disturb- 
ance. In  some  cases  the  disease  process  goes  no  further  than  the 
formation  of  the  eschar,  which  then  becomes  a  scab,  with  subsidence 
of  the  inflammatory  process,  and  recovery.  In  the  majority  of  cases, 
however,  the  disease  runs  a  regular  course;  if  not  excised  early  the 
bacilli  enter  the  blood  stream  and  the  case  terminates  fatally  from  a 
modified  form  of  the  same  disease  as  occurs  in  cattle,  with  the  excep- 
tion that  the  bacilli  are  not  found  in  large  numbers  in  the  internal 
organs  nor  is  the  spleen  greatly  enlarged.  It  may,  therefore,  be  said 
that  in  man  death  is  largely  due  to  the  absorption  of  toxins  from  the 
local  lesions.  When  the  pustule  is  situated  on  the  extremities  the  per- 
centage of  recoveries  under  treatment  is  large. 

PULMONARY  AND  INTESTINAL  ANTHRAX. 

Pulmonary  anthrax,  or  woolsorters'  disease,  is  caused  by  the  inha- 
lation of  spores  by  those  who  handle  hides  or  other  infected  material. 
The  initial  lesion  is  local  and  is  situated  in  the  lower  trachea.  It 
consists  of  a  swollen  and  hemorrhagic  condition  of  the  mucous  mem- 
brane, with  great  enlargement  of  the  mediastinal  and  bronchial 
glands  and  of  effusions  into  the  pleural  and  pericardial  cavities  and 
the  lungs.  Externally  may  occur  also  cutaneous  edema  over  the 
chest  and  neck  and  inflamed  glands.  This  form  is  rapidly  fatal,  the 
patient  dying  from  suffocation  and  toxemia. 

Intestinal  anthrax  is  also  of  local  origin  and  has  about  the  same 
pathology  and  terminations  as  the  pulmonary  form.  There  is  intense 
inflammation  of  the  intestinal  mucous  membrane  and  involvement  of 
the  neighboring  lymphatic  glands.  Intestinal  infection  takes  place 
when  anthrax-infected  products  are  eaten. 

THE  ANTHRAX   SEASON. 

Anthrax  is  peculiarly  a  disease  which  may  be  said  to  be  a  seasonal 
one;  that  is,  the  disease  makes  its  appearance  with  the  advent  of 
certain  kinds  of  weather.  In  March,  1907,  in  Delaware  we  were 
visited  with  some  exceptionally  warm,  springlike  weather  of  con- 


THE  ANTHBAX   SEASON.  21 

siderable  duration,  and  on  the  22d  of  that  month  the  first  case  oc- 
curred for  that  year,  an  exceptionally  early  start.  In  1908  the  first 
case  occurred  on  May  2.  In  1909  the  first  case  did  not  occur  until 
July  23,  but  the  disease  persisted  until  November  6,  whereas  in  1907 
the  last  case  was  on  September  24,  and  in  1908  on  August  24.  So 
that  we  may  say  the  season  lasts,  in  Delaware,  from  March  to  No- 
vember, being  most  prevalent  in  the  summer  months.  As  the  disease 
is,  generally  speaking,  a  warm-weather  one,  and  as  we  know  that 
the  causative  agent  is  present  throughout  the  year,  there  must  be 
some  conditions  which  come  with  warm  weather  which  predispose 
to  the  onset  of  the  disease,  either  by  affecting  the  receptivity  of  the 
animals  of  by  increasing  the  infective  properties  of  the  germ  of  the 
disease.  Possibly  both  conditions  prevail.  There  can  be  little  doubt 
that  the  resting  stage  of  the  Bacillus  anthracis — the  spore — ^will 
quickly  develop  into  the  bacillar  or  infecting  stage  under  the  influ- 
ence of  heat,  moisture,  and  organic  matter.  These  conditions  pre- 
vail on  the  pasture  in  warm  weather,  and  the  writer  has  demon- 
strated that  the  germ  of  anthrax  will  vegetate  in  a  2  per  cent  hay 
infusion.  Hence,  when  spores  of  anthrax  come  in  contact  with  hay 
infusions  in  pastures,  especially  in  meadows,  there  is  produced  a  viru- 
lent culture  which  will  infect  the  animal  eating  them. 

The  animals  themselves  are  probably  more  susceptible  to  anthrax 
when  turned  out  u]3on  fresh  pastures,  as  the  ingestion  of  rank  grasses, 
which  are  acid  in  reaction,  lessens  the  normal  alkalinity  of  the  blood 
and  thereby  increases  the  susceptibility  to  anthrax.  This  gi-eater 
alkalinity  of  the  blood  in  carnivora  may  be  the  cause  of  their  greater 
resistance  to  the  disease,  while  the  loss  of  alkalinity  of  the  blood  by 
the  herbivora,  and  especially  by  ruminants  such  as  cattle  and  sheep, 
through  the  ingestion  of  such  large  quantities  of  acid  grass,  may 
account  for  their  increased  susceptibility  during  the  grass-growing 
season.  At  all  events  there  are  certain  weather  factors  which  de- 
termine, in  a  measure,  the  onset  of  the  anthrax  season,  and  given  such 
conditions  the  season  can  be  predicted  with  tolerable  certainty.  This 
season  exists  when  we  have  had  a  long,  dry  period,  followed  by  light 
rains  or  infrequent  heavy  rains,  and  then  by  extreme  heat.  This  kind 
of  weather  will  not  only  produce  bacterial  multiplication,  but  will 
cause  a  rapid  growth  of  rank  acid  grasses,  and  these  Avhen  eaten 
will  increase  the  susceptibility  of  animals  to  anthrax  by  decreasing 
the  alkalinity  of  their  blood. 

Anthrax,  then,  may  be  said  to  be  a  pasture  disease,  which  exists 
mostly  in  warm  weather.  If  it  were  a  stable  disease,  why  should  we 
not  have  cases  occurring  in  winter,  and  why  is  it  that  the  disease 
rarely  occurs  in  animals  that  are  not  turned  out  on  pasture?  We 
rarely  see  a  case  of  anthrax  in  a  carriage  horse  that  is  fed  in  the 
stall,  but  it  is  not  of  infrequent  occurrence  in  horses  that  are  turned 


22  ANTHRAX. 

out  to  pasture.  Cases  are  rare  in  city  horses,  but  common  in  the 
farm  horse.  The  city  horse  must  eat  the  same  hay  and  grain,  gener- 
ally speaking,  as  the  farm  horse.  If  it  were  the  hay  and  grain  that 
produce  the  disease,  we  should  find  anthrax  as  common  in  city  stalls 
as  on  the  farm. 

Anthrax  rarely  occurs  in  small  towns.  It  is  distinctly  a  disease 
of  animals  that  graze  on  infected  fields.  A  recent  case  emphasizes 
this.  An  expressman  delivered  some  goods  to  a  farmer  who  lives  1 
mile  from  this  town  (Newark,  Del.).  This  farm  has  anthrax  every 
season.  The  expressman's  horse  must  have  grazed  upon  infected 
soil  while  there,  as  it  was  sick  within  48  hours  with  high  fever  and 
edematous  swellings  at  the  root  of  the  neck  and  down  the  front  legs. 
A  microscopic  examination  of  the  exudate  showed  the  presence  of 
antiirax  bacilli,  and  the  animal  died  in  a  few  hours.  This  animal 
lived  in  the  stable  with  others,  and  was  fed  the  same  feed.  No  case 
has  occurred  since,  and  it  was  the  only  animal  from  that  stable  that 
visited  the  infected  farm.  The  great  importance  of  an  early  diag- 
nosis and  isolation  was  demonstrated  in  this  case.  Within  a  few 
hours  after  a  swelling  was  noticed  anthrax  was  suspected  and  some 
of  the  bloody  subcutaneous  exudate  was  aspirated  and  brought  to  the 
writer  by  the  attending  veterinarian  for  diagnosis.  It  was  thus 
possible  to  remove  the  animal  from  the  stable  to  a  yard  where  other 
animals  could  not  become  infected  long  before  any  infecting  dis- 
charge had  taken  place.  The  animal  was  embalmed,  washed  down 
with  formalin  solution,  as  previously  described  in  this  article,  and 
buried  in  a  six-foot  grave  as  soon  as  the  same  could  be  dug.  The 
stables  have  been  used  uninterruptedly  ever  since  (four  months), 
and  no  fresh  case  of  anthrax  has  occurred. 

PREVALENCE  OF  ANTHRAX  IN  DELAWARE. 

Anthrax  has  been  officially  recognized  as  existing  in  this  State 
(Delaware)  since  August,  1892.  It  is  highly  probable  that  the  disease 
had  its  origin  in  imported  hides  used  in  the  tanneries  at  Wilmington 
for  making  leather.  Large  quantities  of  scraps  accumulate  during  the 
process  of  leather  making,  and  these  were  sold  to  farmers  who  com- 
posted them  and  later  spread  the  infected  material  upon  the  land. 
Since  the  spores  of  anthrax  would  live  and  retain  their  virulence  in 
a  compost  heap,  it  is  easy  to  see  how  the  lands  first  became  infected. 
While  it  is  generally  conceded  that  meadow  pastures  are  most  pro- 
ductive of  the  disease,  the  tillable  lands  were  probably  infected  first, 
as  above  stated.  The  meadow  pastures  have  retained  their  infective 
properties  because  of  their  constant  use  for  grazing,  and  particularly 
because  of  fresh  cases  of  anthrax  occurring  upon  them.  The  higher 
tillable  lands  have,  however,  in  many  ca^es  become  disinfected  b>'  the 
lapse  of  time,  cultivation,  and  drainage. 


PBEVALENCE   OF  ANTHRAX  IN  DE1,AWAEE.  23 

Estimates  made  by  veterinarians  practicing  in  the  State  show  that 
there  are  from  175  to  200  farms  in  Delaware  that  are  permanently 
infected  with  anthrax.  These  farms  are  located,  in  a  general  way, 
in  that  territory  which  drains  into  the  Delaware  River  and  the  Dela- 
ware Bay.  The  infected  territory  comprises  about  one-third  of  the 
total  area  of  the  State. 

While  anthrax  has  been  known  to  exist  in  the  State  only  since 
1892,  it  is  highly  probable  that  the  first  cases  occurred  much  earlier 
and  that  the  disease  is  as  old  as  the  morocco-leather  industry.  The 
originally  infected  territory  must  have  been  the  cultivated  lands 
upon  which  infected  compost  fertilizer  was  spread  by  the  farmer  him- 
self. Animals  dying  on  such  farms  before  the  nature  of  the  disease 
was  known  here  were  either  hauled  out  dead  or  turned  out  while  yet 
alive  upon  the  marshes  to  die.  Doubtless  they  would  in  some  cases 
be  set  adrift  in  the  creeks  and  thus  infect  any  shore  or  marsh  upon 
which  they  drifted.  As  the  marshes  are  always  wet  and  contain 
decaying  vegetation  in  abundance,  the  conditions  for  the  development 
of  the  microbe  of  anthrax  are  perfect,  while  upon  the  dry  cultivated 
lands,  even  though  the  microbe  be  present,  the  conditions  are,  owing 
to  the  comparative  absence  of  water,  much  more  unfavorable  to  the 
multiplication  of  the  causative  agent.  It  is  because  of  these  condi- 
tions that  marsh  pastures  are  regarded  as  being  more  productive  of 
the  disease.  The  wash  water  from  the  tanneries  at  Wilmington  can 
no  doubt  infect  the  streams  into  which  it  flows  and  cause  the  disease 
in  animals  either  grazing  upon  them  or  drinking  from  them. 

Those  farms  and  marshes  now  infected  will  remain  so  for  many 
years,  even  though  no  new  cases  occur  upon  them.  This  liability  to 
outbreaks  of  anthrax  on  a  farm  has  a  depressing  effect,  not  only  upon 
the  value  of  the  land,  but  upon  all  agricultural  operations. 

While  there  has  been  no  epidemic  of  anthrax  in  Delaware  for 
several  years,  and  the  number  of  deaths  from  the  disease  has  been 
exceeded  by  many  ordinary  diseases  of  which  little  notice  is  taken, 
anthrax  must  always  be  considered  a  menace  to  the  agricultural  wel- 
fare of  the  State  because  of  the  ever-present  liability  of  an  outbreak 
which  might  assume  the  proportion  of  a  genuine  epidemic.  This 
liability  should  be  offset  by  the  annual  vaccination  of  every  farm 
animal  in  the  infected  territory.  Even  though  several  years  may 
have  elapsed  since  the  last  case  occurred  on  a  farm,  that  farm  is 
liable  at  any  time  to  be  again  visited  by  the  disease. 


24  ANTHRAX. 

METHODS  OF  PRODUCING  IMMTJNITY  IN  ANIMALS. 
THE    EXISTING    METHOD    OF    DOTJBLJE    VACCINATION     (PASTEUR    METHOD). 

Since  1892  anthrax  has  been  controlled  by  vaccination  by  a  method 
devised  by  Louis  Pasteur.  This  method  consists  in  the  subcutaneous 
injection  of  attenuated  cultures  of  the  bacillus  of  anthrax.  Two  in- 
jections of  varying  degrees  of  strength  are  made  at  an  interval  of  12 
to  14  days.  The  first  injection  consists  of  1  cubic  centimeter  of  a 
culture  that  has  been  incubated  at  42°  to  43°  C.  for  a  sujfiicient  time 
to  decrease  its  virulence  to  a  point  where  it  will  kill  white  mice,  but 
not  guinea  pigs  or  rabbits.  This,  generally  requires  a  period  of  24 
days,  assuming  that  the  culture  was  made  originally  from  a  moist, 
virulent  race  of  anthrax  bacilli.  Such  cultures  are  to  be  made 
directly  from  the  heart's  blood  of  an  animal  that  has  died  of  anthrax 
within  48  hours  after  inoculation.  In  such  blood  we  find  only  non- 
spore-bearing  bacilli,  and  when  these  are  grown  at  42°  to  43°  C.  they 
do  not  at  any  time  produce  spores  while  this  temperature  is  main- 
tained. Contrary  to  the  statements  of  some,  however,  the  bacilli 
promptly  form  spores  when  this  temperature  is  reduced.  These 
spores  do  not  acquire  any  more  virulence,  however,  than  the  parent 
bacilli  which  produced  them. 

The  second  injection  consists  of  a  culture  similarly  made  and 
incubated  at  42°  to  43°  C.  for  a  period  of  12  to  18  days,  or  one 
whose  virulence  has  been  reduced  to  a  point  where  it  will  not  kill 
rabbits  but  will  kill  white  mice  and  guinea  pigs,  the  latter  in  3  or  4 
days.  The  author's  experience  has  shown  that  there  is  no  hard  and 
fast  line  in  the  number  of  days  that  a  culture  of  a  given  race  of 
anthrax  bacilli  must  be  attenuated.  Much  depends  upon  the  re- 
sistance of  the  bacillus,  the  character  of  the  culture  medium,  the 
exactness  of  the  temperature  of  the  incubator,  and  the  natural  re- 
sistance of  the  animals  used  in  testing  the  vaccines.  A^Tien  a  culture 
of  proper  strength  has  been  obtained  and  is  properly  transferred 
to  fresh  media  about  once  a  month  and  kept  in  a  cool  place  where 
it  will  not  evaporate,  it  may  be  used  indefinitely  as  a  stock  culture 
for  inoculating  a  liquid  medium,  which  constitutes  the  vaccine.  AVhen 
such  attenuated  cultures  are  inoculated  into  animals,  a  very  mild 
and  clinically  unnoticeable  attack  of  anthrax  is  produced,  which 
confers  an  active  immunity  which  persists  throughout  the  anthrax 
season.    The  inoculation  must  be  repeated  the  following  season. 

Certain  precautions  are  necessary  in  the  application  of  anthrax 
vaccine.  Assuming  that  the  vaccine  has  been  properly  prepared,  it 
is  the  duty  of  the  veterinarian  to  ascertain  that  anthrax  is  not 
already  existing  in  the  animals  he  is  about  to  vaccinate.  As  a  pre- 
caution, when  there  is  reason  to  suspect  that  anthrax  may  already 


METHODS   OF   PRODUCING  IMMUNITY.  25 

be  existing,  no  animal  showing  a  fever  should  be  vaccinated,  as  the 
disease  may  be  carried  from  the  already  infected  animal  to  others 
upon  the  point  of  the  inoculating  needle.  Again,  in  order  to  prevent 
abscess  in  horses  and  mules  by  introducing  under  the  skin  strepto- 
cocci or  staphylococci  by  the  point  of  the  needle,  the  place  of  injec- 
tion should  be  disinfected  of.  these  microbes.  The  anthrax  bacillus 
can  not  produce  pus,  but  the  germs  usually  found  upon  the  skin 
are  to  be  held  responsible  when  abscess  occurs  from  vaccination. 
The  writer  has  been  in  the  habit  of  dipping  the  point  of  the  needle 
into  strong  carbolic  acid  contained  in  a  small  vial  which  may  be 
conveniently  carried  in  the  side  pocket  at  the  time  of  vaccinating. 
None  of  this  acid  can  enter  the  needle  and  kill  the  vaccine  germs, 
as  it  is  already  filled  with  the  vaccine,  and  none  can  be  introduced 
under  the  skin,  as  it  is  all  removed  from  the  needle  in  its  passage 
through  the  skin,  and  this  in  turn  disinfects  the  wound  made.  While 
every  veterinarian  knows  that  hypodermic  injections  of  medicine 
are  daily  made  with  a  minimum  of  abscess  production,  it  must  be 
recognized  that  the  conditions  are  not  similar,  and  hence  it  is  deemed 
highly  advisable  that  these  precautions  be  taken,  especially  in  horses 
and  mules. 

There  is  usuallj^  little  or  no  swelling  at  the  point  operated  upon. 
Where  abscess  occurs  the  operator  is  to  blame.  It  means  that  pus- 
])roducing  germs  have  been  carried  in  upon  the  needle  and  that  either 
the  needle  or  the  skin  was  not  disinfected.  Abscess  is  more  likely  to 
occur  in  horses  and  mules  than  in  other  animals. 

It  has  been  the  practice  in  Delaware  to  continue  the  vaccinated 
animals  at  their  usual  work.  We  have  no  data  upon  the  subject  show- 
ing this  is  unwise,  but  it  is  believed  that  the  animals  should  be  shielded 
as  much  as  possible  from  excessive  work  and  from  extremes  of  heat 
or  cold  or  from  chilling  rains.  We  advise  our  vaccinators,  who  con- 
sist of  regular,  practicing  veterinarians  designated  by  the  governor 
upon  the  recommendation  of  the  board  of  agriculture,  to  destroj^  all 
opened  bottles  of  vaccine  that  remain  unused  at  the  end  of  a  day's 
work,  as  it  will  certainly  become  contaminated  and  be  spoiled  if  kept 
overnight  at  ordinary  temperatures.  The  vaccine  is  dispensed  in 
oO-dose  bottles  hermetically  sealed  and  distinctively  labeled,  so  there 
can  be  no  mistake  made  in  using  it. 

EFFECTIVENESS  OF  THE  METHOD. 

The  results  of  vaccination  have  been  as  good,  as  shown  by  our  sta- 
tistics, as  those  obtained  by  the  use  of  any  other  biological  product. 
That  the  method  is  entirely  safe  is  shown  by  the  fact  that  only  one 
dangerous  swelling  has  been  brought  to  our  notice  in  three  years' 
experience  in  Delaware.    Every  year  there  are  instances  where,  for 


26  ANTHRAX. 

some  reason,  animals  that  have  been  vaccinated  die  from  anthrax, 
showing  that  they  were  not  protected  by  the  vaccine.  No  doubt  some 
of  these  failures  may  be  due  to  the  animal  being  missed  during  vacci- 
nating. In  other  cases  the  failure  may  be  due  to  the  vaccine  needle 
not  properly  puncturing  the  skin,  so  that  the  vaccine  falls  upon  the 
ground.  These  accidents  are  readily  brought  about  when  the  animals 
are  unruly.  On  the  other  hand,  the  writer  has  observed  a  case  of 
anthrax  that  occurred  in  vaccinated  animals — that  is,  where  it  was 
positively  certain  that  the  vaccine  was  properly  prepared  and  active 
and  had  been  properly  applied. 

Some  cases  of  nonprotection  by  vaccination  can  be  explained  by 
the  fact  that  the  vaccine  itself  was  inert,  and  therefore  nonprotec- 
tive.  Cultures  made  from  such  vaccine  failed  to  grow.  The  vaccine 
would,  of  course,  fail  in  the  usual  physiological  test  for  an  anthrax 
vaccine,  and,  of  course,  would  fail  to  protect  the  animal  against 
anthrax.  I'he  writer  has  known  of  instances  where  for  various  rea- 
sons an  animal  would  escape  vaccination,  and  that  animal  would  be 
the  only  one  to  die  on  that  farm.  Hence  he  feels  warranted  in  highly 
commending  vaccination  as  the  most  important  means  of  combating 
this  terrible  scourge.  In  France,  where  vaccination  is  most  popular, 
and  where  statistics  are  reliable  because  of  governmental  control,  the 
death  rate  before  vaccination  was  adopted  was,  in  cattle  5  per  cent, 
and  in  sheep  10  per  cent.  After  vaccination  was  adopted  the  losses 
were  reduced  to  0.34  per  cent  in  cattle  and  0.94  per  cent  in  sheep.  In 
Delaware  the  losses  in  1907  in  horses  and  cattle  that  were  vaccinated 
were  0.32  per  cent. 

Vaccination  should  be  practiced  every  spring,  at  least  a  month  be- 
fore it  is  time  to  turn  the  animals  out  to  pasture,  as  a  month  is  re- 
quired for  the  production  of  immunity.  This  vaccinating  should  not 
be  optional  with  the  owner,  as  at  present,  but  should  be  compulsory, 
the  State  assuming  the  risk  of  loss  from  the  use  of  the  vaccine,  but 
not  in  those  cases  where  it  can  be  shown  the  animal  died  not  from 
vaccination  but  from  a  natural  infection,  owing  to  failure  of  being 
protected  by  the  vaccine,  or  from  other  causes. 

A  TEST  OF  PASTEtJB  VACCINES. 

Along  with  the  investigation  of  anthrax  for  the  purpose  of  dis- 
covering new  methods  of  treatment  and  prevention  of  the  disease, 
and  for  studying  the  biology  of  the  causative  organism  and  the  gen- 
eral sanitary  aspects  of  the  subject,  it  was  decided  to  test  the  efficacy 
of  freshly  prepared  Pasteur  vaccines.  It  is  believed  that  some  of  the 
bad  results  that  have  been  reported  as  following  the  use  of  Pasteur 
vaccine  is  due  to  carelessness  on  the  part  of  those  preparing  and  using 
it.    As  there  is  no  visible  difference  in  the  appearance  of  the  two 


METHODS   OF   PRODUCING  IMMUNITY.  27 

vaccines,  Nos.  1  and  2,  it  is  an  easy  matter  to  get  the  bottles  mixed 
or  improperly  labeled.  If  the  labels  should  come  off  the  bottles,  the 
vaccinator  would  have  no  guide  as  to  which  vaccine  he  was  using. 
The  writer  has  purchased  anthrax  vaccines  on  the  open  market  and 
has  found  some  that  were  wholly  inert,  and  others  that  were  too 
strong  when  tested  in  the  usual  way. 

For  the  purpose  of  gaining  such  information  as  is  possible  from 
the  practical  experiences  of  practicing  veterinarians  who  vaccinate 
against  anthrax  in  Delaware,  the  writer  undertook  to  prepare  Pasteur 
vaccines  for  the  State  board  of  agriculture,  which  were  to  be  used  as 
soon  as  possible  after  they  had  reached  destination.  The  cultures  con- 
stituting the  vaccines  were  grown  in  50-dose  bottles,  each  bottle  con- 
taining 50  c.  c.  of  bouillon  which  had  been  inoculated  24  hours  pre- 
viously with  the  vaccinal  germs.  The  official  vaccinator  was  fur- 
nished first  with  No.  1  vaccine.  In  12  days  he  was  shipped  the  No.  2 
vaccine,  for  use  on  the  same  animals.  Under  this  plan  it  was  impos- 
sible for  the  bottles  of  vaccine  to  get  mixed,  even  though  the  labels 
came  off. 

The  plan  of  using  fresh  vaccine  in  which  the  germ  is  still  in  the 
bacillar  stage  was  continued  with  good  results  for  two  seasons.  For 
the  last  two  years  the  plan  of  using  vaccines  that  had  been  incubated 
for  four  or  five  days,  or  until  the  bacilli  had  spored,  has  been  tried. 
This  latter  method  represents  the  condition  of  vaccine  as  put  on  the 
market  by  commercial  houses,  and  is  a  suspension  of  spores  instead 
of  bacilli.  According  to  the  experiences  of  these  two  seasons,  we 
have  reason  to  believe  that  the  vaccine  will  remain  active  for  several 
months,  and  such  cultures  may  be  prepared  several  months  in  ad- 
vance of  their  use,  provided  the  incubation  is  carried  to  a  point 
where  all  growth  ceases,  or  spores  form.  These  spores  inherit  just 
that  degree  of  strength  possessed  by  their  progenitors,  and  do  not 
change  except  with  a  considerable  lapse  of  time,  possibly  a  year,  if 
kept  under  favorable  conditions.  It  is  highly  important  that  anae- 
robic conditions  be  not  established  in  the  bottles,  as  the  bacilli  will  sink 
to  the  bottom  and  will  not  spore  when  grown  anaerobically.  To  pre- 
vent this,  it  is  important  that  the  culture  medium  be  made  to  reabsorb 
the  air  which  has  been  driven  out  of  it  during  sterilization  before  being 
inoculated  with  the  vaccine  germs.  In  preserving  stock  cultures  of 
the  vaccine  it  is  very  important  that  they  be  incubated  for  several 
days  before  being  taken  out  of  the  incubator.  If  they  are  removed 
before  spores  have  formed,  the  more  vulnerable  bacilli  may  succumb 
to  existing  unfavorable  conditions.  To  these  conditions  may  be 
ascribed  the  death  of  cultures  of  anthrax  vaccine,  and  the  loss  of 
virulence  of  former  virulent  cultures  of  anthrax  bacilli,  which  some- 
times occur  during  the  course  of  laboratory  work. 


28  ANTHRAX. 

EXPERIMENTS  WITH  VABIOUS  SUBSTANCES  TO  TEST  IMMUNIZING 

POWERS. 

Experiments  upon  the  disease  have  been  carried  on  uninterruptedly 
for  the  past  three  years.  These  had  for  their  object  the  preparation 
of  substances  in  the  laboratory  and  in  living  animals  which  could 
be  used  in  combating  anthrax  by  acting  as  antitoxins  or  bactericides, 
or  as  vaccines. 

Realizing  the  great  importance  and  economy  in  producing  pro- 
tective and  curative  substances  in  the  laboratory  over  the  necessarily 
expensive  methods  when  employing  animals  for  the  same  purposes, 
efforts  have  been  made  along  the  line  of  producing  various  culture 
products  to  be  used  in  combating  anthrax. 

Pyocyanase,  made  after  the  method  of  Emmerich  and  Loew,  gave 
some  good  results  when  tested  upon  rabbits,  but  failed  upon  sheep. 

Anthraxase,  prepared  by  the  writer  after  the  same  general  method 
used  in  producing  pyocyanase,  was  without  protective  or  curative 
properties,  although  it  produced  high  fever  when  injected  into  rabbits 
and  sheep  subcutaneously. 

Anthraxoin,  consisting  of  a  turbid  suspension  of  dead  anthrax 
bacilli,  was  apparently  useless  in  protecting  sheep  against  anthrax 
when  used  similarly  to  the  Pasteur  vaccine. 

A  single  vaccine,  having  for  its  object  the  production  of  immunity 
in  two  weeks  and  thus  cutting  down  the  necessary  period  by  one-half, 
was  made  by  incubating  a  virulent  bacillus  for  about  18  days  at  42° 
to  43°  C.  Such  a  culture  will  kill  guinea  pigs  in  about  a  week,  and 
in  strength  it  thus  holds  a  position  between  the  two  vaccines  of 
Pasteur.  With  it  sheep  were  vaccinated  and  after  12  days  withstood 
an  otherwise  fatal  infection  with  virulent  bacilli.  In  some  cases, 
however,  the  immunity  was  not  sufficiently  strong,  as  was  evidenced 
by  the  death,  now  and  then,  of  a  sheep  when  tested  with  virulent 
bacilli. 

The  preparation  and  use  of  the  various  substances,  together  with 
the  results  of  the  experimental  work,  are  described  in  the  following 
pages. 

ANTHRAXIN. 

Anthraxin  was  made  similarly  to  tuberculin  and  mallein.  The 
cultures  of  anthrax  were  grown  for  10  days  with  daily  shaking  in 
glycerinated,  peptonized  bouillon,  the  glycerin  being  used  in  4  per 
cent  strength.  The  cultures  were  then  sterilized  by  boiling,  filtered 
through  Berkefeld  filters,  and  then  evaporated  to  one-tenth  of  the 
original  volume.  A  sirupy  liquid,  much  resembling  tuberculin,  re- 
sulted. It  was  tested  on  rabbits  and  sheep  for  immunizing  proper- 
ties, as  shown  in  Table  1,  on  the  following  page. 


EXPERIMENTS'  WITH   ANTHRAXIN. 
Table  1. — Experiments  with  anthraxin. 


29 


Date. 

Injections. 

Temperature. 

Animal. 

Anthraxin. 

Virulent 
culture 
anthrax. 

Result  and  remarks. 

Rabbit  1      ... 

1907. 
July  10 

1  c.  0.  subcutane- 
ous. 

'F. 

July  17, 10  a.  m 

105,  3  p.  m 

104,10a.  m... 

103,  3  p.  m 

Normal 

104 

July  18 

do 

July  19 

July  24, 11  a.  m. . . 
July  24, 1.30  p.m.. 

1  c.  c.  subcutane- 
ous. 

105 

July  24, 5.30  p.  m.. 

105 

Normal 

July  31 ,  9  a.  m 

0. 05  c.  c. 

Aug.  2,  9  a.  m 

Dead  fr»m  anthrax. 

Sheep  1 

Aug.  27, 1p.m.... 
Aug.  27,  6  p.  m 

5  c.  c.  subcutane- 
ous. 

Sheep  blowing,  head 

down. 
Sheep  seems  well. 

Aug.  28,  9  a.  m 

Sept.  3,  9  a.  m 

Sept.  10,  9  a.  m. . . 

5  c.  c.  subcutane- 
ous. 

0. 25  c.  c. 

Sept.  12,  9  a.  m. . . 

Dead  from  amthrax. 

Sheep  2 

Aug.  27,1p.m.... 
Aug.  27,  6  p.  m 

5  c.  c.  subcutane- 
ous. 

Sheep  blowing,  head 

down. 
Seems  well. 

Aug.  28,  9  a.  m     . 

Sept.  3,9  a.  m 

Sept.  10,9  a.  m 

5  c.  c.  subcutane- 
ous. 
do 

Sept.  23,  9  a.  m 

0. 25  c.  c. 

Sept.  25,  9  a.  m 

Dead  from  anthrax. 

Sheep  3 

Aug.  37,9  a.  m 

5  c.  c.  subcutane- 
ous. 

Blowing;  no  fever. 

Sept.  3,  9  a.  m 

Sept.  5 

5  c.  c.  subcutane- 
ous. 

Diarrhea. 

Sept.  10,9  a.m... 
Sept.  15,  9  a.  m. . . 

5  c.  c.  subcutane- 
ous. 

Well. 

0. 25  c.  c. 

Sept.  17,  9  a.  m.. . 

Dead  from  anthrax. 

Sheep  4 

Aug.  27,  9  a.  m 

Aug.  11,  6  p.  m 

5  c.  c.  subcutane- 
ous. 

Blowing;  no  fever. 

Sept.  13,9  a.m... 

Sept.  10,9  a.m... 
Sept.  15,  9a.  m.. . 

5  c.  c.  subcutane- 
ous. 
do 

Diarrhea  for  several 

days. 

0. 25  c.  c. 

Sept.  17,  9  a.  m.. . 

Dead  from  anthrax. 

Sheep  5  and  C. . . 

Sept.  3,  9  a.  m 

Sept.  0 

5  c.  c.  subcutane- 
ous. 

Seemed  well. 

Sept.  10,9a.  m... 
Oct.  7,  9  a.  m 

5  c.  c.  subcutane- 
ous. 

0.25C.C. 

Oct.  9,  9  a.  m. . 

Both  dead  from  an 

thrax. 

From  the  foregoing  experiments  we  gather  that  anthraxin  was 
possessed  of  no  immunizing  properties  whatever. 


30 


ANTHRAX. 


PYOCYANASE. 


Pyocyanase,  the  next  substance  experimented  with,  was  prepared 
as  follows:  Large  flasks  of  medium  were  inoculated  with  Bacillus 
pyocyaneus  and  grown  at  37°  C.  until  a  ropy  condition  was  produced, 
which  required  three  weeks,  the  flasks  meanwhile  being  shaken  dail3\ 
The  composition  of  the  medium  was  as  follows:  Peptone,  0.5  per 
cent;  glycerin,  0.1  per  cent;  dipotassium  phosphate,  0.1  per  cent; 
magnesium  sulphate,  0.01  per  cent;  sodium  chlorid,  0.3  per  cent; 
sodium  bicarbonate,  0.1  per  cent;  in  distilled  water  (synthetic  medium 
of  Emmerich  and  Loew).  AVhen  growth  had  ceased  in  this  medium 
the  growths  from  a  number  of  agar-agar  cultures  of  the  same  organ- 
ism were  added  and  the  whole  was  thoroughly  shaken.  The  culture 
medium  was  then  neutralized  with  dilute  hydrochloric  acid.  Car- 
bolic acid  was  then  added  to  0.2  per  cent  strength  as  a  preservative. 
The  liquid  was  then  evaporated  down  to  one-tenth  of  its  original 
volume  at  ordinary  room  temperature  by  being  placed  in  pie  plates. 
Then  the  liquid  was  dialyzed  for  24  hours  in  running  water,  filtered 
through  Berkefeld  filters,  and  0.2  per  cent  carbolic  acid  was  again 
added  to  replace  that  which  was  dialyzed  out.  The  resulting  liquid, 
pyocyanase,  has  a  dark  coffee  color  and  a  pungent  odor.  The  experi- 
ments to  test  its  immunizing  and  curative  properties  upon  guinea 
pigs,  rabbits,  and  sheep  are  recorded  in  Tables  2  and  3. 

Table  2. — Experiments  with  unfiltercd  pyocyanase. 


Date. 

Injections. 

Temi>er- 
ature. 

Animal. 

Pyocyanase. 

virulent  24- 

liour  culture 

anthrax. 

Result  and  remarks. 

Babbit  1 

1907. 
Dec.  9,9  a.  m 

Dec.  10,  9  a.  m 

Dec.  11,  9  a.  m 

6  c.  c.  sub- 
cutaneous 


4  c.  c.  sub- 
cutaneous. 

'F. 

0.2  c.c.  sub- 
cutaneous. 

Rabbit    dead.    Liver    no- 

Guinea  pig  1 

3  c.  c.  sub- 
cutaneous 

li  c.  c.  sub- 
cutaneous 

2  c.  c.  sub- 
cutaneous 

3  c.  c.  sub- 
cutaneous 

• 

crosed,  spleen  enlarged; 
anthrax  bacilli  at  local 
lesion,  but  none  found  in 
blood  or  in  liver  and 
spleen;  probably  died  from 
pyocyanase  poisoning. 

Dec.  17, 10  a.m... 
Dec.  18,  10  a.m... 
Dec.  18, 1.30  p.m.. 

Dec.  16, 10  a.m... 
Dec.  17, 10  a.  m... 

0.1  c.  c.  sub- 
cutaneous 

Died  during  night.    No  ba- 

Guinea pig  2 

cilli  in  blood,  but  few  at 
local  lesion;  probably  died 
from  pyocvanase  poison- 
ing. 

0.1  c.  c.  sub- 
cutaneous 

EXPERIMENTS   WITH   UNFILTEEED  PYOCYANASE. 
Table  2. — Experiments  with  unfiltered  pyocyanase — Continued. 


31 


Date. 

Injections. 

Temper- 
attu-e. 

Animal. 

Pyocyanase. 

Virulent  24- 
hour  culture 
antlirax. 

Result  and  remarks. 

Guinea  pig 2 ... 

1907. 
Dec.  18,  9  a.  m 

Dec.  19,9  a.  m 

IJ  c.  c.  sub- 
cutaneous. 

•  F. 

Sick. 

Cliloroformed.    No  bacilli  in 

Guinea  pig  3 

Dec.  19,  9  a.  m 

Dec.  20, 10  a.  m... 
Dec.  21,  3  p.m.... 

1908. 
Jan.  2,  9  a.  m 

3  c.  c.  sub- 
cutaneous. 
do 

blood,  but  a  few  at  local 
l^lon;  pyocyanase  poi- 
soning. 

IJ  c.  c.  sub- 
cutaneous. 

::::::::::::::::::::: 

0.  Ice.  sub- 
cutaneous. 

Jan.  4  9  a.  m 

Eabbit2 

Jan.  13  9  a.  m 

Jan.  14,  9  a.  m 

Jan.  15>  9  a.  m. ... 

6  c.  c.  sub- 
cutaneous 
...  do 

0.2  c.  c.  sub- 
cutaneous 

in  blood;  another  guinea 
pig  treated  similarly  died 
of  anthrax  on  third  day 
after  inoculation. 

103 
102 

Jan.  16,  9  a.  m 

Pound    dead    of    anthrax. 

Rabbits 

Jan.  13, 9  a.  m 

Jan.  14, 9  a.  m 

Jan.  15,9  a.  m 

Jan.  18,  9  a.  m 

6  c.  c.  sub- 
cutaneous. 

6  c.  c.  sub- 
cutaneous. 

Bacilli  in  blood. 

.... 

0.3  c.  c.  sub- 
cutaneous 

Edema  at  points  of  injection 

Normal. 

of  pyocyanase,  no  fever. 

Animal  apparently  well. 

Jan.  25,  9  a.  m 

Animal  dies  from  anthrax 

Rabbit  4 

Jan.  13  9  a.  m 

Jan.  14.  9  a.  m 

Jan.  15,  9  a.  m 

Jan.  16,  9  a.  m 

6  c.  c.  sub- 
cutaneous. 

do 

do 

0.2  c.  c.  sub- 
cutaneous 

on  eleventh  day.  Bacilli 
in  blood. 

Normal. 
...do 

Dead  from   anthrax.     An- 

Rabbits  

Feb.  3, 10  a.  m 

Feb.  4, 10  a.  m 

Feb.  6, 10  a.  m 

J  c.  c.  sub- 
cutaneous. 
do 

..  do 

other  rabbit  treated  simi- 
larly died  of  anthrax  on 
third  day  also;  bacilli  in 
blood. 

0.3  c.  c.  sub- 
cutaneous. 

102.8 

Slight  swelling  at  point 
where  pyocyanase  was  In- 
jected. 

Feb,  8, 10  a.  m 

•         1 

Animal  dead  from  anthrax, 

Rabbit  6 

Feb.  3, 10a.m.... 
Feb.  4.  10  a.  m 

Feb.  5  10  a.m.... 
Feb.  7, 10  a.  m 

Ice.  sub- 
cutaneous. 
do.... 

having  lived  four  days. 

n  'A  ^  f^   snh- 

Con.siderable     reaction     at 

cutaneous. 
do 

point  of  injections. 

1 

Rabbit  dead  from  anthrax 

Rabbit? 

Feb.  3, 10  a.m.... 
Feb.  4,  10  a.m.... 
Feb.  5, 10  a.  m 

2  c.  c.  sub- 
cutaneous. 
da- 

on  third  day. 

0.3  e.e.  sub- 
cutaneous. 

103 
103 

Small  reaction  from  injec- 
tions. 

Feb.  8, 10  a.  m i 

Animal  dead  from  anthrax, 

1 

having  lived  four  days. 

32  ANTHRAX. 

Table  2. — Experiments  with  unflHered  pyocyanase — Contiuued. 


Date. 

Injections. 

Temper- 
ature. 

Animal. 

Pyocyanase. 

Virulent  24- 
hour  culture 
anthrax. 

Result  and  remarks. 

Rabbits 

1908. 
Feb.  3,  10  a.  m 

Feb.  4,  10  a.  m 

• 

Feb.  7, 10  a.  m 

4  c.  c.  sub- 
cutaneous. 

1  c.  c.  sub- 
cutaneous. 

'F. 

0.3  c.  c.  sub- 
cutaneous. 

loas 

Some  reaction  from  injec- 
tions. Abscess  formed  at 
point  of  injection. 

Rabbit  9 

Feb.  3, 10  a.  m 

Feb.  4, 10  a.  m 

Feb.  5, 10  a.  m 

5  a  a  sub- 
eutaneoos. 

2  c.  c.  sub- 
cutaneous. 

0.2  c.  c.  sub- 
cutaneous. 

Severe  reaction  at  point  of 
injection. 

101 
105 

Feb.  8,  10  a.  m 

Animal  dead  from  anthrax 

on  fourth  day. 

The  foregoing  experiments  were  made  with  unfiltered  pyocyanase. 
The  dead  bacilli  produce  abscesses  at  points  of  injection  and  fre- 
quently disease  of  the  liver,  which  contribvited  to  death  from  anthrax. 

Table  3. — Experiments  ivith  filtered  pyocyanase. 


Date. 

Injections. 

Tem- 
perature. 

Animal. 

Pyocyanase. 

Virulent  24- 
hour  culture 
of  anthrax. 

Result  and  remarks. 

Sheep  1     

1909. 
Jan.  4, 10  a.  m 

Jan.  4, 1  p.  m 

10  c.  c.  sul> 
cutaneous. 

'F. 
103.6 

105 
106 

103.6 
103 

Jan.  4,  3p.  m 

Jan.  5,  9  a.  m 

10  c.  c.  sub- 
cutaneous. 

0.1  c.  c.  sub- 
cutaneous. 

Jan.  5,  4  p.  m 

Off  feed. 

Jan.  5,  7  p.  m 

Dead  from  pyocyanase  poi- 
sonmg. 

Sheep  2 

Jan.  6,  9  a.  m 

Jan.  6, 12m 

3  c.  c.  sub- 
cutaneous. 

103 

105.5 
106.9 
104 

Jan.  7, 10  a.  m 

Jan.  7, 11  a.  m 

1  c.  c.  sub- 
cutaneous. 

Respirations  jerky. 

Jan.  21,  3p.  m 

Jan.  22,  9  a.  m 

Jan.  22,  3  p.  m 

Jan.  23,  9  a.  m 

Jan.  23,  3  p.  m 

Jan.  25,  9  a.  m 

5  c.  c.  sub- 
cutaneous. 

4  c.  c.  sul> 
cutaneous. 

2  c.  c.  sub- 
cutaneous. 

3  c.  c.  sub- 
cutaneous. 

2  c.  c.  sub- 
cutaneous. 

103.5 

102.5 

102.5 

103 

102 
102 

1 

0.1  c.  c.  sub- 
cutaneous. 

Jan.  26,  12  m 

Jft".  2*<|  10  a,  tn 

Dead  from  anthrax,  having 

Sheep  3 

Jan.  7, 10  a.  m 

Jap.  7,  4  p.  "1 . , , 

2  c.  c.  sub- 
cutaneous. 

102 
107.2 

101 
101 

lived  5  days. 

Animal  does  not  seem  sick 

Jan.  13,  9  a.  m 

Jan.  13, 3  p.  m 

2  c.  c.  sub- 
cutaneous. 

5  c.  c.  sub- 
cutaneous. 

even  though  temperature 
is  high. 

0.1  c.  c.  sub- 
cutaneous. 

EXPERIMENTS   WITH   FILTERED   PYOCYANASE.  33 

Table  3.— -Experiments  with  filtered  pyoeyanase — Continued. 


Date. 

Injections. 

Tem- 
perature. 

Animal. 

Pyoeyanase. 

Virulent  24- 
hour  culture 
of  anthrax. 

Result  and  remarlts. 

Sheep  3 

1909. 
Jan.  14,  9  a.  m 

Jan.  14  1.30  p.  m.. 

2  c.  c.  sub- 
cutaneous. 

°F. 
102.5 

102.5 
104.5 

Jan.  15, 10  a.m.... 
Jan. 16 

2  c.  c.  sub- 
cutaneous. 

Anthrax  fever  setting  in. 

Dead  from  anthrax  (3  days;. 

Sheep  4 

Jan.  8, 10  a.  m 

Jan  8  1  p.  m 

1  c.  c.  sub- 
cutaneous. 

101 
102 

Jan.  8,  3  p.  m 

1  c.  c.  sub- 
cutaneous. 
do 

0.1  c.  c.  sub- 
cutaneous. 

103 

103.5 

105.5 

103.3 
103 

Jan.  10,  4.30  p.m.. 
Jan.  11,  9  a.  in 

do 

Dead  two  hours  later  of  an 

Rabbit  10 

1908 
Mar.  5,  9  a.  m 

Mar.  6,  9  a.  m 

Mar.  7,  9  a.  m 

0.05  c.c.  sub- 
cutaneous. 

0.5  c.  c.  sub- 
cutaneous. 

....do...... 

thrax  (3  days). 

Mar.  8,  9  a.  m 

0.2  c.  c.  sul> 
cutaneous. 

Mar.  9,  9  a.  m 

Mar.  13,  9  a.  m 

0.5  c.  c.  sub- 
cutaneous. 

102.8 

Rabbit  well. 

Mar.  18,  9  a.  m 

Found  dead  of  anthrax  (10 

Rabbit  11 

Mar.  5,  9  a.  m 

Mar.  6,  9  a.  m 

1  c.  c.  subcu- 
taneous, 
do 

days). 

103 

Mar.  7,  9  a.  m 

Mar.  8,  9  a.  m 

do 

0.2  c.  c.  sut)- 
cutaneous. 

103 

101.5 

102.6 

103.7 

103 

103 

Mar.  10,  9  a.  m 

Mar.  18,  9  a.  m 

Mar.  21,  9  a.  m 

Mar.  23,  9  a.  m 

Mar.  24,  9  a.  m 

1.5  c.  c.  sub- 
cutaneous. 

3  c.  c.  sub- 
cutaneous. 

5  c.  c.  sul> 
cutaneous. 

Animal  sicii. 

Animal  much  better. 

Apr.  12,  9  a.  m 

Much  emaciated  and  swol- 

Apr. 13,  9  a.  m ... . 

len  along  Ijelly. 
Dead  from  anthrax.    Post- 

Rabbit 12 

Mar.  5,  9  a.  m 

Mar.  6,  9  a.  m 

1.5  c.  c.  sub- 
cutaneous. 
do 

102 

102 
103 

102.8 
102.8 
103 

104.3 

mortem  e  -x  a  m  i  n  a  tion 
showed  linear  abscess  in 
liver;  repeated  larger  doses 
of  pyoeyanase  probably 
caused  animal  to  succumb 
to  anthrax  (lived  5  weeks); 
cheek  rabbit  died  in  48 
hours  after  inoculation. 

Mar.  7,  9  a.  m 

Mar.  8,  9  a.  m 

do 

...    do 

0.2  c.  c.  sub- 
cutaneous. 

Mar.  18,9  a.m.... 
Mar.  20,  9  a.  m 

Mar.  24,  9  a.  m 

Apr.  12,  9  a.  m 

Apr.  14,  9  a.  m 

do 

3  c.  c.  sul)- 
cutaneous. 

5  c.  c.  sub- 
cutaneous. 

Animal  has  kept  well. 

0.1c.  c.  sub- 
cutaneous. 

Animal    found    dead    from 

Rabbit  13 

Mar.  18,  9  a.  m 

Mar.  18,  2  p.  m 

5  c.  c.  sub- 
cutaneous. 
do 

anthrax,  showing  Immu- 
nity was  only  passive 
(lived  24  days). 

0.2  c.  c.  sub- 
cutaneous. 

34  ANTHRAX. 

Table  3. — Experiments  with  filtered  pyocyanase — Continued. 


Date. 

lajections. 

Tem- 
perature. 

Animal. 

Pyocyanase. 

Virulent  24- 
hour  culture 
of  anthrax. 

Result  and  remarks. 

Babbit  13 

1908. 
Mar.  18,  4  p.  m 

'F. 
104 
104 
101 

Mar.  19,  9  a.  m 

Mar.  20,  9  a.  m 

Mar.  28 

5  c.  c.  sub- 
cutaneous. 

Rabbit  14 

Nov.  24,9  a.  m 

Nov.  24,  3p.m... 
Dec.  4,  9  a.  m 

1  c.  c.  sub- 
cutaneous. 
do 

do 

thrax.  Period  of  incuba- 
tion lengtliened  to  10  days 
by  pyocyanase;  two  more 
rabbits  similarly  treated 
reacted  the  same,  and 
lived  10  days  and  7  days, 
respectively. 

Simultaneous  Injections  of 

0.2  e.  c.  sub- 
cutaneous. 

103 

"ase"  and  bacilli. 

Dec.  5 

Dec.  16 

3  c.  c.  sub- 
cutaneous. 

Feb.  4 

Found  dead  from  anthrax. 

Rabbit  15 

Nov.  24, 11  a.  m. . . 
Nov.  24,3.  pm... 
Dec.  4,  9  a.  m 

2  c.  e.  sub- 
cutaneous. 
do 

do 

Lived  21  days. 

0.2  e.  c.  sul)- 
cutaneous. 

104 

Some  swelling  which  disap- 
pears in  2  days. 
Animal  has  remained  well 

Dec.  5 

Dec.  16 

Feb.  4 

3  c.  c.  sub- 
cutaneous. 

up  to  Feb.  24,  when  it  was 

killed  by  dog  (lived  69 
days). 

Rabbit  16 

Nov.  24,11a.m... 
Nov.  24,  3  p.m.... 
Dec.  4,  9  a.  m 

3  c.  c.  sub- 
cutaneous. 
do 

do ; 

0.2  c.  c.  sub- 
cutaneous. 

104 

Animal  remains  well. 

Dec.  5,  9  a.  m 

104.8 

Swollen  from  "ase"  injec- 
tion. 

Dec.  16,9  a.  m 

Feb.  4 

3  c.  c.  sub- 
cutaneous. 

Animal  has  remained  well 

Rabbit  17 

Nov.  24,  11a.m... 
Nov.  24,  3  p.  m 

4  c.  c.  sub- 
cutaneous. 
....do 

up  to  Feb.  4,  when  it  was 
killed  by  dog  (lived  69 
days).  No  lesions  on  post- 
mortem; check  rabbit  died 
in  48  hours  after  inoaila- 
tlon. 

Simultaneous  Injection. 

0.2  c.  c.  sub- 
cutaneous. 

104 

Chloroformed  to  locate  posi- 
tion and  stage  of  develoi> 
ment  of  bacilli.  No  spores 
or  bacilli  found  by  micro- 
scope or  culture  in  local 
lesion,  liver,  spleen,  or 
peritoneal  cavity.  Check 
rabbit  died  In  48  hours 
after  inoculation. 

From  the  foregoing  experiments  with  pyocyanase  it  will  be  seen 
that  when  given  in  proper  dose  and  simultaneously  with  virulent 
anthrax  bacilli,  the  period  of  inoculation  is  greatly  lengthened  in 
rabbits.  Sheep,  however,  seem  to  be  very  susceptible  to  poisoning  by 
pyocyanase,  and  no  immunity  is  conferred  upon  them  by  its  use. 


EXPERIMENTS  WITH   ANTHEAXASE. 


35 


ANTHRAXASE. 

Anthraxase  was  made  in  about  the  same  manner  as  pyocyanase, 
except  that  the  Bacillus  anthracis  was  substituted  for  Bacillus  pyo- 
cyaneus.  The  tests  of  this  substance  include  cultures  grown  in  ordi- 
nary bouillon,  cultures  grown  in  Emmerich  and  Loew's  medium,  and 
experiments  with  precipitated  anthraxase.  The  details  are  given  in 
Tables  4,  5,  and  6. 


Table  4. — Experiments  tvith  anthraxase — Cultures  grown  in  ordinary  bouillon. 


Date. 

Injections. 

Temper- 
ature. 

Animal. 

Anthraxase. 

Anthrax 
culture. 

Results  and  remarks. 

Rabbit  18 

1908. 
Mar.  17,  9  a.  m 

Mar.  18,  9  a.  m 

Mar.  19,  9  a.  m 

Mar.  20,  9  a.  m 

Mar.  21,  9  a.  m 

Mar.  23,9  a.  m 

0.75  c.c.  sub- 
cutaneous. 

0.50c.c.sub- 

cutaneous. 

do 

3  c.c.  subcu- 
taneous. 

5e.c.  subcu- 
taneous. 
do 

°F. 
101 

101.5 

102 

102.4 

107 

0. 2  c.  c.  sub- 
cutaneous. 



Animal  very  sick. 
Dead  from  anthrax  (lived 
5  days). 

Mar.  25,  9  a.  m  . 

Rabbit  19 

Mar.  17,  9  a.  in 

Mar.  18,  9  a.  m 

1.5  c.c.  sub- 
cutaneous. 
do 

100.5 

102 
102.  C 

104.6 

Swollen  lymphatics. 

Mar.  19,9  a.  m 

Mar.  20,9  a.  m 

Mar.  21,  9  a.  ni 

2  c.c.  subcu- 

taneous. 

3  c.  c.  subcu- 

taneous. 

0.2  c.  c.  sub- 
cutaneous. 

Dead  from  anthrax  on  sec- 

Rabbit 20 

Mar.  17,  9  a.  m 

Mar.  18,  9  a.  m . . . 

1  c.c.  subcu- 
taneous. 
..  .do 

101 
101.5 

ond  day. 

Mar.  19,  9  a.  ra 

Mar.  20,  9  a.  m 

0.2  c.  c.  sub- 
cutaneous. 

104. 1 
103 

Anthrax  fever. 

Mar.  21,9  a.  m 

Mar.  22,  9  a.  m 

3  c.c. subcu- 
taneous. 

Rabbit  21 

Mar.  23,9  a.m.... 
Mar.  24,  9  a.  m 

0.5  c.  c.  sub- 
cutaneous. 
do 

103 

104 
104 

day. 
Animal  pregnant. 

Mar.  20,  9  a.  m 

do 

Mar.  23,  9  a.  m 

Mar.  24,  9  a.  m 

5  c.  c.  intra- 
venously. 

3  c.  c.  subcu- 
taneous. 

2  c.  c.  subcu- 
taneous. 
do 

0.2  c.  c  sub- 
cutaneous. 

Animal  died  from  shock. 

Rabbit  22 

104. 5 

104 
103. 1 

Mar.  20,  9  a.  m 

do 

Mar.  27,  9  a.  m 

5  c.  c.  intr.v- 
venously. 

3  c.  c.  subcu- 
taneous. 

0.2  c.  c.  sub- 
cutaneous. 

102.9 
102.4 

Mar.  28,  9  a.  m 

Mar.  30,9  a.  m 

4  c.  c.  subcu- 
taneous. 

Died  of  anthrax  on  fourth 

Rabbit  23 

Mar.  23,  9  a.  m 

Mar.  24,  9  a.  m 

4  c.c.  subcu- 
taneous. 
do 

105 
100. 5 

day. 
Animal  excited. 

Mar.  2.i,  9  a.  m .   . . 

Mar.  20,  9  a.  m 

5  c.  c.  subcu- 
taneous. 

0.2  c.  c.  sub- 
cutaneous. 

103.4 

36  ANTHRAX. 

Table  4. — Experiments  with  anthraxase,  etc. — Continued. 


Date. 

Injections. 

Temper- 
ature. 

Animal. 

Anthraxase. 

Anthrax 
culture. 

Results  and  remarks. 

Rabbit  23 

1908. 
Mar.  28.  9  a.  m 

Mar.  30,  9  a.  m.... 

4  c.  c.  subcu- 
taneous. 

°F. 

• 

Died  of  anthrax  on  third 

Rabbit  24 

Mar.  23,9a.  m 

Mar.  24,  9  a.  m.... 

6  c.c.  subcu- 
taneous. 
do 

104.1 

105 
103.9 

day. 
Animal  pregnant. 

Mar.  25,  9  a.  ni 

Mar.  26,  9  a.  in 

do 

Mar.  28,  9  a.  m 

Mar.  30,  9  a.  in 

6  c.  e.  intra- 
venously. 

3  c.  c.  subcu- 
taneous. 

4c.  c.  subcu- 
taneous. 

0.2  e.  c.  sub- 
cutaneous. 

103.0 

Rabbit  25 

Mar.  25,9  a.  m 

Mar.  26, 9  a.  in 

r 

do 

3  c.  c.  intra- 
venously. 

5  c.  0.  Intra- 
venously. 

3  c.c.  subcu- 
taneous. 

0.5  c.  c.  sub- 
cutaneous. 
do 

0.1c.  c.  sub- 
cutaneous. 

103 
103.8 

day. 
Animal  pregnant. 

Much  depressed  by  the  in- 
jections and  aborted  at  4 
p.m.    (Control  for  this  se- 
ries dies  in  48  hours  after  in- 
oculation with  the  same 
culture.) 

Rabbit  26 

Apr.  7,9a.  m..... 
Apr.  8,  9  a.  m 

102 
103 

Apr.  9,9  a.  m 

Apr.  10,  9  a.  m 

do 

do 

0.1  c.  c.  sub- 
cutaneous. 

Apr.  11,  9  a.  m 

Apr.  11,  4  p.  m 

Apr.  12,  9  a.  m 

2  c.c.  subcu- 
taneous. 

3  c.c.  subcu- 

taneous. 

105 
105.3 

Dead  from  anthrax  on  third 

day. 

Tabt.k   5. 


-Ex  peri  in  ruts   irifJi    nnthrnxase.     Cultures   provu   in    Emmerleh    and 
Loeir/H  medium. 


Date. 

Injections. 

Temper- 
ature. 

Animal. 

Anthraxase. 

.\nthrax  cul- 
ture. 

Results  and  remarks. 

Rabbit  27 

1908. 
Apr.  7, 10  a.  m 

Apr.  9,10  a.  m 

Apr.  10, 10  a.  m. . . 
Apr.  11, 10a.  m... 

Apr.  11, 4  p.  m 

0.5  c.  c.  sut)- 
cutaneous. 

<i°-.- 

do 

1  c.  c.  sub- 
cutaneous. 

3  c.  c.  sub- 
cutaneous. 

°F. 

102 

102 

102 
102.3 

102.  5 

103 

0.2  c.  c.  sub- 
cutiiueous. 

Much  .swollen. 

Apr.  13,  4  p.  m. . 

Dead  from  anthrax  on  fifth 

Rabbit  28 

Apr.  15, 10a.  m... 
Apr.  16, 10a.  m... 

0.25  c.  c.  sub- 
cutaneous. 
do 

day. 

100.8 
99.5 
99.8 

101 

102.8 

Apr.  17,  It)  a.  m... 
Apr.  18, 10a.  m... 
Apr.  25, 10a.  m... 

.\pr.  26, 10  a.  m . . . 

-Vpr.  27, 10  a.  m . . . 

do 

do 

do 

O.SOc.c  sub- 
cutaneous. 

0.2  c.  c.  sul)- 
cutaneous. 

Dies  of  anthrax  in  2  days. 

EXPERIMENTS   WITH    ANTHRAXASE. 


37 


The  following  experiments  on  rabbits  and  sheep  were  made  with 
precipitated  anthraxase.  Absolute  alcohol,  9  volumes  to  1  of  anthrax- 
ase  solution,  was  employed  as  the  precipitant.  The  resulting  yellow, 
gummy  precipitate  was  readily  soluble  in  water,  to  which  0.2  per  cent 
strength  of  carbolic  acid  was  added. 

Table  6. — Experiments  ivith  precipitated  anthraxase. 


Dile. 

Injections. 

Temper- 
ature. 

Animal. 

Anthraxase. 

Anthrax 
culture. 

Result  and  remarks. 

Rabbit  ''O 

1908. 
Apr.  15,9  a.  m 

0.5  c.  c.  sub- 
cutaneous. 
do 

°F. 
101 

101 
101 
101 

Apr.l7,9a.m 

do 

do 

.. 

Apr.  25, 9  a.  m 

Apr.  26,9  a.  m 

do 

do 

0.01  c.c.  sub- 
cutaneous. 

101.8 
102 

Apr.27,9a.  m 

Apr.  27,5  p.  m 

Apr.  28,9  a.  m 

do 

5  c.  0.  sub- 
cutaneous. 

Ciieclc  rabbit  dies. 

• 

103 
103 

Apr.  29,9  a,  in 

Apr.  30    .          .   . 

Animal  seems  perfectly 
well. 

Nov.  5,9  a.  m 

Nov.  9,9  a.  m 

0.1  c.  c.  sub- 
cutaneous. 

immunity. 

Rabbit  30.. 

May  2, 10  a.  m 

1  c.  c.  sub- 
cutaneous. 
do 

103.3 

102.5 
103 

102 

104.5 
105 

104 
100.3 

102. 3 

101 
102 

104.8 

102.  5 

101 
102 
102.  5 

May  2,  4p.  m 

May  4,  10  a.  in 

do 

3  c.  c.  sub- 
cutaneous. 

0.1  c.  c.  .sub- 
cutaneous. 

May  5,  11..30a.  m.. 
May  5,  4  p.  m 

2  c.  c.  sul)- 
cutaneous. 

May  6,  10  a.  m. . . 

Dead  of  anthrax  at  4  p.  m. 
on  fifth  day. 

Rabbit  31 

May  2,  10  a.  m 

May  2,  2  p.  m.. 

1  c.  c.  sub- 
cutaneous. 
do. 

May  2,  4  p.  m 

May  4, 10  a.  in . .   . 

do 

0.1  c.  c.  sul)- 
cutaneous. 

Anthra.x  fever.    Check  dies 

May  5, 10  a.  in 

May  5,  4  p.  m 

2  c.  c.  sub- 
cutaneous. 

to-day. 

May  6, 10  a.  m. . . . 

May  7,  10  a.  ni 

May  8,  10  a.  m. . 

Animal  turned  out  in  yard. 

May  14, 10  a.  m  . . . 

Sheep  5 

June  29,  10  a.  in... 
June  29,  12  in 

5  c.  c.  sub- 
cutaneous. 
do 

mortem  showed  death 
from  anthrax  and  cocci- 
diosis  (lived  12  days). 

June  29,  2  p.  in 

Juno  29,  4  p.  m 

June  30,  lOa.m  ... 
Juno  .30, 12  m... 

.do..  .. 

do 

do 

0.1  c.  e.  sub- 
cutaneous. 

.   .do 

June  .30,  2p.  m 

Juno  .30,  4  p.  in 

Julv2,  9  a.  m... 

do 

...do 

do 

10.5.  S 
104.3 
105.8 

July  2,  10  a.  m 

do 

July  2, 12  m 

.   .do  .... 

T>ied  of  anthrax  at  2  )>.  ni. 

Shocp  0 

June  29,  10  a.  in... 
Jime29,  12  in 

.do..    . 

on  fourth  day. 

do 

Juiio29,  2p.  in.... 

do 

38  ANTHRAX. 

Table  6. — Experiments  with  precipitated  anthraxasc — Continued. 


Date. 

Injections. 

Temper- 
ature. 

Animal. 

Anthraxase. 

Anthrax 
culture. 

Result  and  remarks. 

Sheep  6 

1908. 
June  29,  4p.  m 

June  30, 10  a.m... 
Juuo30,  12  m 

5  0.  c.  sub- 
cutaneous. 
do 

0.1  c.  c.  sub- 
cutaneous. 

"F. 

do 

June  30,  2  p.  m 

do 

•. 

June  30,  4p.m 

do 

July  2,  8  a.  ni 

do 

102.2 

102 
102 
102.8 
103 

Check  guinea  pig  dies  in  2 
days. 

July  2,  10  a.  m 

July  2,  12  m 

do 

do 

July  2,  3p.  m 

do 

July  2,  7  p.  m 

do 

July  4, 10  a.  m 

do 

Sheep  7 

July  28,  10  a.m... 
July  28,  12  m 

do 

sixth  day. 

do 

July  28,  2p.  m 

do 

July  28,  4p.m 

July  29, 10  a.m... 
July  29, 12  m 

do 

do 

do 

0.1c.  e.  sub- 
cutaneous. 

102.3 

102 

102.3 

102.6 

106 

106 

106 

106.2 

July  29,  2  p.  m 

July  29,  4p.m.... 
July  30, 10  a.  m... 
July  30,  12  m 

do 

do 

do 

...  .do 

July  30,  2  p.  m 

do 

July  30,  4  p.  m 

July31, 10a.  m  ... 

do 

Found  dead  of  anthrax  on 

fourth  day  after  inocula- 
tion of  tHst  dose. 

ANTHRAXOIN. 

Anthraxoin  was  prepared  and  experimented  with  as  follows:  It 
consists  es.sentially  of  a  suspension,  in  carbolized  normal  salt  solution, 
of  dead,  sporeless,  anthrax  bacilli.  It  was  produced  by  inoculatint?, 
from  a  fresh  anthrax  carcass,  bottles  that  had  been  filled  completely 
with  nutrient  bouillon  and  plugged  with  hardest  paraffin  stoppers. 
In  this  way  none  but  sporeless  bacilli  were  introduced,  and  under  the 
existing  anaerobic  conditions  no  spores  could  form.  After  all  growth 
had  cea.sed,  at  35°  C,  these  cultures  were  completely  immersed  in  a 
water  bath  maintained  at  55°  C.  for  one  hour,  which  was  sufficient 
to  devitalize  the  bacilli  but  not  to  destroy  any  antibodies  they  might 
contain.  The  dead  bacilli  were  separated  by  filtration  from  the 
liquid  in  which  they  had  grown,  and  those  adhering  to  the  Berkefeld 
filter  were  washed  off  in  the  requisite  amount  of  carbolized  normal 
salt  .solution.  The  suspension  experimented  with  consisted  of  the 
bacilli  that  grew  in  2,500  c.  c.  of  bouillon,  su.spended  in  50  c.  c.  of 
carbolized  normal  sodium  chlorid  solution.  The  details  are  given  in 
Table  7. 


EXPERIMENTS  WITH   ANTHRAXOIN. 
Table  7. — Experiments  with  anthraxoin. 


39 


Date. 

Injections. 

Temper- 
ature. 

Animal. 

Anthraxoin. 

Anthrax  cul- 
ture. 

Result  and  remarks. 

Sheep  8. . 

1908. 
Apr.  24,  10  a.  m . . . 

May  8,  10  a.  m 

May  20,  10  a.  m . . . 

1  c.  c.  subcu- 
taneous. 

2  c.  c.  subcu- 
taneous. 

op 

0.25  c.  c.  sub- 
cutaneous. 

May  22, 10  a.  m.. 

Sheep  dies  of  anthrax  in  2 
days. 

Sheep  9... 

Apr.  24,  10  a.  m... 
Mays,  10  a.  m 

2  c.  c.  subcu- 
taneous. 

0.25  c.  c.  sub- 
cutaneous. 

May  9,  10  a.  m 

102.2 
103 
101.5 
101.5 

May  9,  4  p.  m 

May  10,  10  a.  m 

May  11,10  a.  m 

Sheep  10 

Apr.  24,  10  a.  m... 

May  8,  10  a.  m 

May  20,10  a.  m.. 

0.5  c.  c.  sub- 
cutaneous. 

1  c.  c.  subcu- 
taneous. 

0.25  c.  c.  sub- 
cutaneous. 

Sheep  dies  of  anthrax  in  3 
days. 

Sheep  11 

May  11, 10  a.  m 

May  24, 10  a.  m 

1  c.  c.  subcu- 
taneous. 

0.25  c.  c.  snh- 
cutaneous. 

Sheep  dies  of  anthrax  in  2 
days. 

Sheep  12 

May  11, 10  a.  m 

May  20, 10  a.  m 

1  c.  c.  subcu- 
taneous. 

0.25  c.  c.  sub- 
cutaneous. 

Sheep  dies  of  anthrax  in  3 

Sheep  13 

May  11, 10  a.  m 

May  23, 10  a.  m 

June  8,  10  a.  m 

1  c.  c.  subcu- 
taneous. 

5  Q.  c.  subcu- 
taneous. 

days. 

0.25  c.  c.  sub- 
cutaneous. 

June  10,  10  a.  m... 

Sheep  dead  from  anthrax  in 
2  days. 

Sheep  14 

May  11,10  a.m.... 

May  23, 10  a.  m 

June  8,  10  a.  m 

1  c.  c.  subcu- 
taneous. 

5  c.  c.  subcu- 
taneous. 

0.25  c.  c.  sub- 
cutaneous. 

June  12,  10  a.  m . . . 

Sheep  dead  from  anthrax  in 
4  days. 

Sheep  15 

Apr.  20,  9  a.  in 

Apr.  26,  12  m 

10  c.  c.  sub- 
cutaneous. 
do 

103 

104 
104 
104 

103 

103 
103.5 
103.5 
104 

103. 5 
104 

Apr.  26,  2  p.  m.. 

.     .do 

Apr.  26,  4p.  m 

Apr.  27,  9  a.  m 

Apr.  27,  12  m 

do 

12  c.  0.  sub- 
cutaneous. 
do 

0.25  c.  c.  sub- 
cutaneous. 

Apr.  27,  2  p.  m 

do 

..   ..do 

Apr.  28,  9  a.  m 

Apr.  28,  2  p.  m 

10  c.  c.  sub- 
cutaneous, 
do 

Apr.  28,  4p.  m 

.  ..do 

Apr.  29,  10  a.  m. . 

Sheep  dead  from  anthrax  in 

Rabbit  32 

Mar.  8,  10  a.  m 

Mar.  8, 1  p.  m 

0.5  c.  c.  sub- 
cutaneous. 

3  days. 

104.1 
105.4 
101.8 

Mar.  8,  4.30  p.  m.. 

Mar.  9,  9».  m 

0.2  c.  c.  sub- 
cutaneous. 

40 


ANTHEAX. 
Table  7. — Experiments  with  anthraxoin — Continued. 


.  Date. 

Injections. 

Temper- 
ature. 

Animal. 

Anthraxoin- 

Anthrax  cul- 
ture 

Result  and  remarks. 

Rabbit  32 

1908. 
Mar.  10,  9  a.  m 

105.8 

Mar.  11,  9  a.  m 

Guinea  pig 

Mar.  8,  10  a.  m 

Mar.  9, 10  a.  ni 

0.5  c.  c.  sub- 
cutaneous. 

days. 

0.2  c.  c.  sub- 
cutaneous. 

Mar.  11, 10  a.  in... 

Rabbit  33 

Mar.  16,  9  a.  m 

Mar.  16,  11  a.  m... 

0.1  e.  c.  sub- 
cutaneous. 

101 

102.2 
102.5 
104.3 
101.5 
102 

103.5 

days. 

Mar.  16,  12  m 

- 

Mar.  10,4.30  p.m.. 

Mar.  17,  9  a.  m 

Mar.  26,  9  a.  m 

Mar.  26,  4  p.m.... 

0.1  c.  c.  sub- 
cutaneous. 

Apr.  6,  9  a.  m 

0.01  c.  c.  sub- 
cutaneous. 

Apr.  8,  9  a.  m 

Rabbit  34 

Mar.  16,  9  a.  m 

Mar.  16, 11  a.  m... 

0.2  c.  c.  sub- 
cutaneous. 

102 

103.6 

104.5 

105.3 

102 

102.5 

105 

in  2  days. 

Mar.  16, 12  m 

Mar.  16, 1..30  p.  m.. 

Rabbit  stupid. 

Mar.  26,  9  a.  m 

Mar.  26,  2p.  m 

0.2  c.  c.  sul> 
cutaneous. 

' 

Apr.  6,  9  a.  m 

0.01  c.  e.  sub- 
cutaneous. 

Apr.  8,  9  a.  m 

Rabbit  35 

Mar.  16,  9  a.  m 

Mar.  16, 11  a.  m... 

0.3  c.  c.  sub- 
cutaneous. 

102 

103.2 

103.5 

104 

105 

102.5 

102 

104.5 

in  2  days. 

Mar.  16,  12  m 

Skin  swollen  at  injection. 

Mar.  16,  2p.  m 

Mar.  16,  4  p.  m 

Much  swollen  from  injection. 

Mar.  17,  9  a.  m 

Swelling  practically  gone. 

Mar.  26,  9  a.  m 

Mar.  26,  4  p.  m 

0.3  c.  c.  sub- 
cutaneous. 

0.01  c.  0.  sub- 
cutaneous. 

Apr.  6,  9  a.  m 

Dead  from  anthrax  in  2  days. 

A    COMMERCIAL  VACCINE   IN    PILL   FORM. 

A  commercial  vaccine  which,  according  to  the  makers,  consists  of 
dead  anthrax  organisms  in  pill  form  was  also  tested.  These  small 
pills  are  placed  under  the  skin  by  means  of  a  trocar  and  are  claimed 
by  the  makers  to  produce  immunity  to  anthrax.  Microscopic  exami- 
nation, as  well  as  cultural  and  animal  experiments,  show  that  the 
claims  of  the  makers,  in  so  far  as  the  vaccine  being  dead  and  harm- 
less is  concerned,  are  true.  One  can  easily  see  with  the  microscope 
that  these  little  pills  consist  of  dead  anthrax  bacilli  and  their  spores 


EXPERIMENTS   WITH   COMMERCIAL  VACCINE. 


41 


held  together  in  pill  form  by  a  proper  excipient.  The  writer  was 
unable,  however,  to  verify  the  claim  that  they  produce  any  im- 
munity, as  is  shown  in  Table  2.  A  rabbit  succumbed  in  six  days,  but 
as  it  is  very  rarely  that  a  rabbit  can  be  immunized  by  a  vaccine,  a 
sheep,  which  animal  is  easily  protected,  was  also  employed,  with 
negative  results. 

Table  8. — Experimetits  with  commercial  vaccine  in  pill  form. 


Date. 

Injections. 

Temper- 
ature. 

Animal. 

Vaccine. 

Anthrax 
culture. 

Result  and  remarks. 

Rabbit  36 

1908. 
Apr.  7,  9  a.  m 

Apr.  8,  9  a.  in 

Apr.  9,  4  p.  m 

1  pill,  sub- 
cutaneous. 

1    pill   (dis- 
solved), 
subcuta 
neous. 
do 

"F. 

do 

0.01  C.C.  sub- 
cutaneous. 

Apr.  10,  4  p.  m 

Apr.  11,  4  p.  m 

1  pill,  sub- 
cutaneous. 

104 
104.3 

Apr.  13,  4  p.  in 

Apr.  15,  4  p.  m 

Dead    from   anthrax    in   6 

Sheep  16 

Oct.  17,  9  a.  m 

Nov.  2,  9  a.  m 

1   pill  witli 
trocar. 

days. 

0.1  c.  c.  sub- 
cutaneous. 

Nov.  4,  9  a.  in 

Sheep  dead  from  anthrax 

in  48  hours. 

PREPARATION    OF    AN    EFFECTIVE    SINGLE   VACCINE. 

The  single  vaccine,  like  Pasteur  vaccine,  consists  of  cultures  of 
attenuated  anthrax  bacilli,  the  only  difference  being  in  the  degree 
of  attenuation  and  that,  as  its  name  implies,  it  is  applied  only  once, 
thus  requiring  a  shorter  time  and  only  one  handling  of  the  animals. 

To  prepare  such  a  vaccine  or  attenuated  culture,  the  virulence  of 
an  already  virulent  culture  of  anthrax  was  exalted  by  passage 
through  very  young  animals.  A  strain  can  in  this  way  be  pro- 
duced which  will  kill  a  sucking  rabbit  in  24  hours.  From  the  heart'ti 
blood  of  such  a  carcass  tubes  of  bouillon  are  inoculated  immediately 
after  death.  These  cultures  are  then  incubated  at  42°  to  43°  C. 
for  varying  periods  of  time — from  12  to  18  days.  On  the  twelfth 
day  a  subculture  is  made  from  one  of  the  tubes  and  cultivated  at 
3.5°  C.  On  the  thirteenth  and  succeeding  days  subcultures  are  made 
from  the  remaining  tubes  until  the  series  has  been  completed.  These 
six  subcultures  of  attenuated  bacilli  are  now  tested  on  guinea  pigs 
and  rabbits,  and  on  sheep  if  possible. 


42  ANTHRAX. 

More  dependence  is  to  be  placed  upon  the  animal  test  than  upon 
the  number  of  days  the  attenuation  process  is  carried  on.  A  cul- 
ture of  proper  strength  is  generally  obtained  from  tubes  that  have 
been  attenuated  for  about  16  days.  The  proper  culture  will  be  one 
which  will  not  kill  rabbits,  but  which  will  kill  a  majority  of  guinea 
pigs  in  a  delayed  period,  say  5  or  6  days.  Such  an  animal  should 
show  no  swelling  at  the  point  of  inoculation,  and  the  bacilli  should 
be  found  only  sparingly  in  the  internal  organs.  When  shaken  in 
cultures  such  a  vaccine  shows  a  homogeneous  clouding  of  the  medium ; 
no  flocculi  persisting,  as  occurs  in  virulent  cultures. 

As  rabbits  and  guinea  pigs  can  not  be  made  immune  to  anthrax 
by  vaccination  of  this  kind,  these  animals  are  only  useful  in  testing 
the  pathogenesis  of  the  cultures.  For  testing  the  immunizing  prop- 
erty, sheep,  which  are  easily  immunized,  were  employed.  Quite  a 
number  of  sheep  were  used  in  the  experiment  to  produce  a  safe, 
efficient  single  vaccine,  and  as  regards  the  failures  along  this  line, 
it  is  only  necessary  to  say  that  they  were  entirely  due  to  improper 
attenuation  of  the  cultures.  When  the  proper  attenuation  was  reached 
there  was  no  difficulty  in  immunizing  animals  by  a  single  vaccina- 
tion. This  was  effective  against  a  subsequent,  otherwise  mortal  dose 
of  virulent  anthrax  bacilli,  as  the  following  experiments  will  show. 

TESTS  OF  THE  SINGLE  VACCINE. 
EXPEBIMENT  No.   1. 

On  April  15,  1909,  a  visibly  pregnant  cow  was  given  subcuta- 
neously  1  c.  c.  of  single  vaccine.  She  showed  no  ill  effects  whatever 
from  the  injection.  On  May  1  the  animal  was  inoculated  with  0.2 
c.  c.  of  virulent  anthrax  bacilli  from  a  24-hour  culture  which  killed 
a  check  rabbit  and  a  check  cow  in  48  hours.  On  May  19  the  cow 
dropped  a  fully  developed  calf  which  when  found  was  dead,  lying 
with  its  head  bent  under  the  shoulder.  Supposing  that  the  calf  had 
been  asphyxiated,  it  and  the  membranes  were  buried.  However,  a 
platinum  loop  full  of  the  discharge  was  plated  and  a  pure  culture 
of  60,000  anthrax  bacilli  was  obtained.  A  guinea  pig  inoculated 
with  a  culture  made  from  one  of  these  colonies  died  of  anthrax  in 
48  hours.  The  cow  remained  well,  and  subsequent  daily  cultivations 
from  vaginal  discharges  showed  no  anthrax,  bacilli. 

This  experiment  was  extremely  valuable,  not  only  as  showing  that 
the  cow  had  been  immunized  by  a  single  vaccination,  but  also  for 
showing  not  only  that  anthrax  can  be  communicated  not  only  to  the 
fetus,  but  that  it  can  be  thus  communicated  by  an  immune  mother. 
It  also  showed  that  anthrax  bacilli  may  persist  for  at  least  18  days 
in  the  body  of  an  immune  animal.  When  the  cow  was  destroyed  for 
various  reasons  on  May  25  she  was  in  perfect  health,  and  cultures 


SERUM   FOE   PRODUCING   IMMEDIATE   IMMUNITY.  43 

and  inoculations  made  with  her  blood  showed  no  anthrax  bacilli 
present.  It  is  to  be  regretted  that  this  animal  could  not  have  been 
kept  for  further  observations  and  experiments. 

Experiment  No.  2. 

On  September  14,  1909,  two  sheep  were  vaccinated  with  single 
vaccine,  each  receiving  1  c.  c,  and  showed  no  sickness  therefrom.  On 
September  28  each  sheep  was  inoculated  subcutaneously  with  0.2  c.  c. 
of  a  culture  of  anthrax  bacilli  whose  virulence  had  been  proven  on  a 
rabbit  in  the  same  experiment.  The  sheep  at  no  time  showed  any 
sickness.  On  November  2  both  sheep  were  again  tested  with  virulent 
bacilli  and  showed  no  sickness. 

Experiment  No.  3. 

On  November  12,  1909,  three  sheep  were  vaccinated  with  1  c.  c.  of 
single  vaccine  and  showed  no  sickness  therefrom.  On  November  27 
one  of  the  animals  was  tested  with  0.2  c.  c.  of  virulent  bacilli,  and 
on  December  1  the  other  two  were  similarly  tested.  They  at  no 
time  showed  any  sickness.  These  three  sheep,  together  with  the  two 
used  in  experiment  No.  2,  were  used  later  in  experiments  to  produce 
an  antibacterial  serum. 

A  SERUM  FOR  PRODUCING  IMMEDIATE  IMMUNITY. 

Although  the  favorable  results  from  vaccination  by  the  Pasteur 
system  have  been  known  for  a  long  time,  and  owing  to  the  cheapness 
of  the  vaccine  it  would  seem  that  there  is  nothing  more  to  be  desired, 
the  length  of  time  required  to  produce  immunity  is  one  drawback  in 
its  use  when  one  is  endeavoring  to  check  an  existing  outbreak  of  the 
disease.  Wlien  vaccination  is  practiced  a  month  in  advance  of  the 
animals  l^eing  turned  out  to  pasture  in  the  spring,  the  system  of 
Pasteur  vaccination  is  the  proper  one  to  use.  In  existing  outbreaks, 
however,  it  is  evident  that  any  system  that  requires  a  month  to  be- 
come protective  leaves  much  to  be  desired,  as  many  animals  could  be- 
come infected  and  die  before  protection  could  be  afforded  them. 

With  the  end  in  view  of  devising  a  method  whereby  an  immediate 
immunity  could  be  established  in  existing  outbreaks  or  where  an  im- 
munity could  be  brought  about  in  a  much  shorter  time  than  has  been 
possible  under  the  old  system  of  vaccination,  the  writer  has  devoted 
a  large  portion  of  his  time  for  the  past  three  years.  As  it  had  been 
conclusively  demonstrated  that  animals  can  be  immunized  by  a  single 
vaccine,  thus  cutting  down  one-half  the  period  necessary  for  immuni- 
zation by  the  old  system  of  Pasteur,  it  was  decided  to  experiment 
upon  the  production  of  an  antianthrax  serum    by    endeavoring    to 


44  ANTHRAX. 

hyperimmunize  sheep  after  the  manner  of  the  production  of  anti- 
hog-cholera  serum,  with  the  exception  that  whereas  in  the  latter  work 
virulent  blood  was  used,  this  was  precluded  in  our  experiments  owing 
to  the  dangers  attending  the  handling  of  large  quantities  of  virulent 
anthrax  blood.  Instead,  the  blood  used  was  that  drawn  from  an 
animal  immune  to  anthrax,  and  not  from  one  sick  with  anthrax. 

Briefly  stated,  the  writer  has  produced  an  antibacterial  serum  by 
highly  immunizing  sheep  through  repeated  inoculations,  first  of  at- 
tenuated anthrax  bacilli,  and  following  these  by  inoculations  of  the 
most  virulent  races  of  the  bacilli  in  increasing  doses  until  the  animal 
would  withstand  with  impunity  fifty  thousand  times  the  minimal 
lethal  dose.  Such  a  serum  will  protect  a  sheep  against  an  otherwise 
mortal  dose  of  bacilli  and  produce  an  immediate  immunity.  It  is, 
therefore,  a  very  valuable  adjunct  in  working  against  the  spread  of 
the  disease  in  existing  outbreaks  where  the  usual  vaccination  is  be- 
ing practiced.  The  serum  will  confer  a  passive  immunity  immedi- 
ately, and  thus  protect  the  animal  against  fatal  infection  until  the 
vaccine  confers  an  active  immunity.  And  should  an  animal  which 
has  been  protected  by  the  serum  become  infected  with  a  virulent 
anthrax  bacillus,  the  results  of  this  infection  will  be  the  production 
of  a  much  stronger  immunity  than  the  vaccine  and  serum  would 
otherwise  confer. 

It  is  evident  that  if  a  single  vaccine,  or  even  a  double  vaccine,  can 
be  used  in  conjunction  with  an  antibacterial  serum  to  produce  im- 
mediate passive  immunity  which  will  persist  until  the  vaccine  has 
had  time  to  bring  about  active  immunity  a  long  step  will  have  been 
made  in  controlling  this  formidable  disease.  The  experiments  with 
this  end  in  view  were  carried  out  upon  sheep  as  follows: 

EXPERIMENTS  WITH  THE  SERUM. 

Experiment  No.  1. 

On  December  15,  1909,  a  sheep  immunized  with  single  vaccine  and 
a  subsequent  inoculation  with  virulent  bacilli  (see  Experiments  Nos. 
2  and  3  with  single  vaccine)  was  bled  from  the  carotid  artery — the 
femoral  artery  being  small  and  deep-seated — by  means  of  a  glass 
cannula  and  rubber  tubing.  ^Vhen  the  blood  had  clotted,  500  c.  c.  of 
serum  were  decanted  and  injected  into  the  inguinal  regions  of  another 
sheep  which  had  been  similarly  immunized.  Microscopic  examina- 
tions and  inoculations  into  guinea  pigs  of  this  blood  showed  it  to  be 
free  of  anthrax  bacilli.  The  injected  sheep  was  lame  in  both  hind 
legs  the  next  day,  but  this  lameness  disappeared  when  the  complete 
absorption  of  the  injected  blood  had  taken  place,  and  no  abscess 
formed.     On  January  12  this  sheep  was  bled  from  the  carotid  artery, 


EXPERIMENTS  WITH   THE  SEEUM.  45 

and  when  the  serum  was  collected  it  was  preserved  by  adding  to  it 
one  part  of  a  5  per  cent  solution  of  carbolic  acid  to  each  nine  parts 
of  serum.  In  making  this  addition  of  carbolic  acid  some  coagulation 
will  occur  if  the  preservative  and  serum  be  not  poured  simultaneously 
into  another  vessel  and  stirred  by  a  helper. 

The  above  serum  was  without  protective  properties.  All  animals 
upon  which  it  was  used  died  of  anthrax,  as  follows : 

On  January  13,  1910,  12  guinea  pigs  received  increasing  doses  of 
serum  from  0.1  up  to  3  c.  c.  and  simultaneously  0.1  c.  c.  of  virulent 
anthrax  culture.     All  of  these  animals  died  of  anthrax  in  48  hours. 

On  January  17, 1910,  the  amounts  of  serum  were  increased  to  4  c.  c, 
5  c.  c,  6  c.  c,  7  c.  c,  8  c.  c,  9  c.  c,  and  10  c.  c,  with  a  simultaneous  dose 
of  0.1  c.  c.  of  virulent  culture.  The  7  animals  thus  tested  also  died  in 
48  houi*s. 

On  F'ebruary  5,  1910,  a  sheep  received  subcutaneously  24  c.  c.  of 
the  serum  and  0.2  c.  c.  of  virulent  culture.  A  control  rabbit  died  in 
48  hours  and  the  sheep  died  of  anthrax  in  three  days. 

On  February  9,  10,  and  11  another  sheep  received  daily  12  c.  c.  of 
the  serum  and  on  the  12th  a  test  dose  of  virulent  bacilli.  This  sheep 
lived  four  days  and  then  died  of  anthrax. 

These  experiments  demonstrated  that  the  modified  hog-cholera 
method  of  serum  production  could  not  be  applied  to  anthrax,  and 
it  was  then  decided  to  try  and  bring  about  hyperimmunization  by 
increasing  doses  of  virulent  bacilli  repeated  over  a  long  period.  The 
two  sheep  used  in  experiment  No.  2  with  single  vaccine  were  em- 
ployed for  this  purpose  as  follows : 

Experiment  No.  2. 

On  September  14,  1909,  two  sheep  were  vaccinated  with  1  c.  c.  of 
single  vaccine.  On  September  28  they  were  given  a  test  dose  of  0.2 
c.  c.  of  virulent  bacilli,  and  remained  well,  while  a  check  rabbit  died 
in  48  hours.  On  November  2  each  sheep  again  received  0.2  c.  c.  of 
virulent  bacilli.  On  March  4,  1910,  each  sheep  received  0.3  c.  c.  of 
virulent  bacilli.  On  March  18  they  received  1  c.  c.  of  virulent  bacilli, 
and  on  April  2  each  sheep  received  5  c.  c.  of  virulent  bacilli.  A 
period  of  about  seven  months  was  thus  consumed. 

On  April  14,  1910,  these  sheep  were  bled  from  the  carotid  artery, 
producing  1,900  c.  c.  of  serum,  to  which  was  added  one  part  of  a  5 
per  cent  solution  of  carbolic  acid  to  each  nine  parts  of  serum  as  a 
preservative.  Prior  to  the  addition  of  the  preservative  tests  for  the 
presence  of  anthrax  bacilli  were  made  upon  guinea  pigs  and  by  plate 
cultures.    None  were  found. 

This  serum  was  used  upon  sheep  in  connection  with  virulent  an- 
thrax bacilli  as  shown  in  Table  9. 


46 


ANTHRAX. 
Table  9. — Experiments  \cith  antianthrax  serum. 


Animal. 


Date. 


Injections. 


Immune  serum. 


Virulent  culture. 


Results  and  remarks. 


Sheep  17 

Rabbit  (control) 
Sheep  18 

Rabbit  (control) 
Sheep  19 

Rabbit(control) 
Sheep  20 

Sheep  21 

Rabbit  (control) 
Sheep  22 

Sheep  23 

Sheep  24 

Sheep  25  (con- 
trol). 

Rabbit(control) 


1910. 
Apr.  29 
May   16 
..do 


5  c.c.  subcutaneous. 


May  21 
June  20 
July  G 
May   21 

May  24 
June  20 
July  6 
May   24 

May  27 
June  20 
July     6 

May  27 
June  20 
July  fi 
June  20 

June  1 
June  18 
July     6 

June  1 
June  18 
July     G 

June  1 
June  18 
July     6 

June  18 


July     6 


10  c.  c.  subcutaneous. 

....do 

....do 


10  c.  c.  subcutaneous. 

....do 

....do 


10c.  c.  subcutaneous. 
do. 


10  c.  c.  subcutaneous. 
do 


5  c.c.  subcutaneous. 
10  c.  c.  subcutaneous. 
do 

5  c.  c.  subcutaneous 
10  c.  c.  subcutaneous. 
do 

5  c.  c.  subcutaneous. 
10  c.  c.  subcutaneou.s 
do 


0. 2  c.  c.  subcutaneous . . . 
.  2  c.  c.  subcutaneous. . . 
.  1  c.  c.  subcutaneous . . . 

.  1  c.  c.  subcutaneous. . . 
.  2  c.  c.  subcutaneous. . . 
.  5  c.  c.  subcutaneous . . . 
.  1  c.  c.  virulent  culture. 

.  1  c.  c.  subcutaneous. . . 
.  1  c.  c.  subcutaneous . . . 
.  5  c.  c.  subcutaneous . . . 
.Ice.  virulent  culture. 

.  5  c.  c.  single  vaccine. . . 
.  1  c.  c.  virulent  culture. 
.See.  virulent  culture. 

.  5  c.  c.  single  vaccine. . . 
.  1  c.  c.  virulent  culture. 
.5  c.  c.  virulent  culture. 
.Ice.  virulent  culture. 

1. 0  c.  c.  single  vaccine . . . 
.  1  e.  c.  virulent  culture. 
.  5  c.  c.  virulent  culture. 

1.0c.  c.  single  vaccine. . . 
.  5  c.  c.  virulent  culture. 
.  5  c.  c.  virulent  culture. 

1. 0  c.  c.  single  vaccine. . . 
.  1  c.  C.  virulent  culture. 
.  5  c.  c.  virulent  culture. 

.  1  c.  c.  virulent  culture. 


,  1  c.  c.  virulent  culture 


Sheep  remains  well. 
Rabbit  dies  in  48  hours. 

Not  sick. 
No  bad  result. 
Remained  well. 
Died  in  48  hours. 


Remained  well. 
Died  in  48  hours. 

Remained  well. ' 

Do. 
Dead  in  48  hours. 

Remained  well. 
Do. 
Do. 


Died  of  anthrax  in  48  hours 
(control  for  sheep  22,  23, 
and  24,  for  inoculations 
June  18). 

Died  of  anthrax  in  48  hours 
(control  for  sheep  22,  23, 
and  24,  for  inoculations  on 
July  6. 


No  doubt  the  period  of  time  occupied  in  hyperimmunizing  the 
sheep  which  produced  this  highly  protective  serum  can  be  very  much 
lessened — probably  one-half.  It  will  be  noted  that  the  time  covered 
was  about  seven  months.  This  was  not  intentional,  but  as  the  sheep 
had  already  been  immunized  for  another  object,  and  not  used,  it  was 
decided  to  carry  out  the  idea  of  greatly  increasing  their  immunity 
by  the  method  followed.  It  will  be  seen  by  referring  to  the  table 
that  an  idle  period  of  about  three  months  elapsed  between  November, 
1909,  and  February,  1910,  which  might  just  as  well  have  been  em- 
ployed for  the  purpose.  It  is  also  possible  that  by  giving  larger 
doses  of  virulent  bacilli  the  immunizing  property  of  the  serum  may 
be  considerably  increased. 

That  the  degree  of  immunity  can  be  established  by  the  number  of 
inoculations  and  the  quantity  of  virulent  culture  used  was  clearly 
shown  in  sheep  Nos.  18,  19,  20,  21,  22,  23,  and  24.     They  had  received 


CONCLUSION.  47 

three  injections  of  serum  and  three  inoculations  with  bacilli,  in  very 
small  doses,  and  while  they  themselves  were  immune  (see  Table  9) 
their  blood,  when  tested  after  slaughter  on  July  18,  did  not  protect 
other  sheep. 

It  is  also  highly  probable  that  sheep  will  produce  a  more  effective 
antianthrax  serum  for  their  own  species,  and  cattle  for  cattle,  horses 
for  horses,  etc. ;  that  is,  the  sera  should  be  homologous.  However,  no 
effort  was  made  to  test  this  principle,  as  the  great  expense  attending 
such  experiments  precluded  their  being  carried  out. 

CONCLUSION. 

The  writer  does  not  advise  the  abandonment  of  the  old  Pasteur 
system  of  vaccination  against  anthrax  when  it  is  practiced  upon  ani- 
mals before  they  are  turned  out  on  the  pastures  in  the  spring  of  the 
year.  When  animals  are  dying,  however,  vaccination  alone  requires 
too  long  a  period  to  protect,  and  it  is  in  these  outbreaks  that  the  anti- 
anthrax  serum  should  be  used  in  conjunction  with  vaccine.  The  ex- 
periments have  shown  that  a  single  vaccine  may  be  used  with  good 
results.  Where  it  is  desired,  however,  the  serum  may  be  used  simul- 
taneously with  the  double  vaccine  or  with  a  single  vaccine. 

o 


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